CN113786391B

CN113786391B - mRNA delivery system and preparation method and application thereof

Info

Publication number: CN113786391B
Application number: CN202111114926.XA
Authority: CN
Inventors: 李斌; 李三朋; 黄逸轩; 李敏; 吴伟刚; 吴嘉彩
Original assignee: Shenzhen Peoples Hospital
Current assignee: Shenzhen Peoples Hospital
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2022-07-26
Anticipated expiration: 2041-09-23
Also published as: CN113786391A; WO2023045036A1

Abstract

The invention provides an mRNA delivery system, a preparation method and application thereof, wherein the mRNA delivery system comprises a carrier and mRNA loaded on the carrier; the carrier comprises lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules; the quaternary ammonium salt lipid-like molecule has a structure shown as a formula I or a formula II; the mRNA delivery system is prepared by a method comprising: mixing quaternary ammonium salt lipid-like molecules with an organic solvent to obtain an organic phase; mixing the organic phase with water to obtain a mixed phase; and mixing the mixed phase with an mRNA solution to obtain an mRNA delivery system. The mRNA delivery system has simple components, can realize high-efficiency and high-specificity lung targeting mRNA delivery by using a single-component or double-component carrier, has excellent stability, good biocompatibility, simplified preparation process and good stability among batches, and lays a foundation for the application of mRNA medicaments in treating lung diseases.

Description

mRNA delivery system and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an mRNA delivery system, and a preparation method and application thereof.

Background

Messenger rna (mrna) is a single-stranded ribonucleic acid that is transcribed from a DNA strand as a template, carries genetic information, and can direct protein synthesis, and it functions at the gene level as one of nucleic acid drugs. The mRNA medicine has the characteristics of short research and development period, high safety, quick synthesis, good immunogenicity and the like, and is widely used in the fields of protein replacement therapy, cancer immunotherapy, vaccines and the like at present. As an important and difficult point of mRNA drug research, mRNA delivery vehicles are advantageous in protecting exogenous mrnas from nuclease degradation, and introducing mrnas that are difficult to enter cells into cells, and then producing functionally active proteins through the body's protein translation system. Therefore, the development of safe and effective mRNA delivery systems is one of the key technologies for mRNA drugs.

For example, CN111467321A discloses an mRNA nucleic acid drug delivery system, which includes lipid nanoparticles for loading one or more mrnas, the lipid nanoparticles are prepared from raw materials including ionizable cationic lipid, phospholipid helper lipid, cholesterol, and phospholipid polyethylene glycol derivatives, and have good and stable intracellular delivery efficiency of mRNA drugs. CN112999360A discloses an application of DMP nanoparticles in mRNA delivery, and provides DMP nanoparticles prepared by mixing DOTAP cationic phospholipid and amphiphilic diblock copolymer mPEG-PCL aiming at the problem that the existing mRNA delivery lacks a high-efficiency and safe carrier, so as to provide a new delivery carrier for the mRNA delivery; the DMP nanoparticles have low cytotoxicity and high transfection efficiency in mRNA delivery, and have good prospects. CN111658782A discloses an mRNA vaccine delivery vehicle, which comprises a lipid molecule a and a lipid molecule B, wherein the lipid molecule a is prepared by the reaction of bromononene and trifluoromethyl octyl-2-ene-1-ol, and the lipid molecule B is synthesized by the reaction of cyclodisinine and trifluoromethyl 1, 2-epoxydodecane; the mRNA vaccine delivery vector is applied to an mRNA vaccine and also comprises stabilizing agents such as dioleoyl phosphatidyl ethanolamine, cholesterol and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000; the mRNA vaccine delivery vector can realize safe expression and activate immune response in vivo, and has better stability, delivery efficiency and safety. From the current research results, there have been clinical application successes of nucleic acid drugs using lipid nanoparticle delivery technology, such as clinical mRNA vaccines BNT162b2 and mRNA-1273, siRNA drugs inpattro, and the like.

However, most of the existing mRNA lipid nano-delivery vectors are complex in components, most of the translated proteins are enriched in the liver, and the problem of specific targeted delivery of other organs besides the liver, such as the lung, is urgently to be solved, so that the therapeutic level of nucleic acid drugs is greatly limited. Therefore, the development of a highly efficient nucleic acid drug delivery system with organ targeting is the focus of research in this field.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an mRNA delivery system and a preparation method and application thereof, and by adopting the lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules as a carrier, the problem of organ specificity of the nucleic acid drug delivery system is solved, the effect of lung targeted delivery is realized, the mRNA is highly specifically expressed in the lung, and a foundation is laid for the treatment of the lung diseases of the mRNA drug.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an mRNA delivery system comprising a vector and mRNA loaded on the vector; the carrier comprises lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules; the quaternary ammonium salt lipid-like molecule has a structure shown in a formula I or a formula II:

wherein R is ₁ 、R ₂ 、R ₃ Each independently selected from any one of C1-C50 straight chain or branched chain alkyl, C2-C50 straight chain or branched chain unsaturated alkyl, C3-C50 alicyclic hydrocarbon group, C2-C50 fat heterocyclic group, C6-C50 aromatic hydrocarbon group or C6-C50 aromatic ring-containing alicyclic hydrocarbon group;

R ₄ ^- is a monovalent anion;

n represents the number of methylene groups and is an integer of 0 to 10, and may be, for example, 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.

The mRNA delivery system is prepared by a method comprising: mixing quaternary ammonium salt lipid-like molecules with an organic solvent to obtain an organic phase; mixing the organic phase with water to obtain a mixed phase; and mixing the mixed phase with an mRNA solution to obtain the mRNA delivery system.

In the mRNA delivery system provided by the invention, the lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules are used as a carrier, so that the problem of organ specificity of the nucleic acid drug delivery system is solved, the efficient and high-specificity lung-targeted mRNA delivery is obtained, the stability is excellent, the biocompatibility is good, the components are simple, the preparation process is simplified, and a foundation is laid for the treatment of the lung diseases by the mRNA drug.

In the present invention, the linear or branched alkyl group of C1 to C50 may be a linear or branched alkyl group such as C2, C5, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45, or C48.

The C2 to C50 straight-chain or branched-chain unsaturated hydrocarbon group may be a straight-chain or branched-chain unsaturated hydrocarbon group such as C3, C5, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45, or C48, and the "unsaturated hydrocarbon group" means a group containing at least one C ≡ C double bond or at least one C ≡ C triple bond.

The C3-C50 alicyclic hydrocarbon group can be C3, C5, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45 or C48, and the like, and the alicyclic hydrocarbon group means a non-aromatic carbocyclic group and comprises a saturated alicyclic hydrocarbon group or an unsaturated alicyclic hydrocarbon group; the saturated alicyclic hydrocarbon group includes monocyclic, polycyclic (fused rings containing 2,3 or 4 rings), spiro, and includes, by way of example and not limitation: cyclohexane, cycloheptane, adamantyl, and the like; the "unsaturated alicyclic hydrocarbon group" means an alicyclic hydrocarbon group containing at least one C ═ C double bond or at least one C ≡ C triple bond, and exemplarily includes but is not limited to: cyclohexenyl, cycloheptenyl, and the like.

The C2 to C50 aliphatic heterocyclic group may be an aliphatic heterocyclic group such as C3, C5, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45, or C48, that is, a group formed by introducing a heteroatom into an alicyclic hydrocarbon group, and the meaning of the alicyclic hydrocarbon group is as described above, and is not described herein again.

The C6 to C50 aromatic hydrocarbon group may be C6, C9, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45, or C48, and the like aromatic hydrocarbon group, and exemplarily includes but is not limited to: phenyl, naphthyl, biphenyl, anthracenyl, phenanthrenyl, and the like.

The above-mentioned C6 to C50 (for example, C6, C9, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45, C48, etc.) includes an aromatic ring-containing alicyclic hydrocarbon group, i.e., a group formed by introducing an aromatic ring to an alicyclic hydrocarbon group, and the meaning of the alicyclic hydrocarbon group is as described above, and is not described herein again.

In the present invention, the heteroatom in the lipoheterocyclic group includes, but is not limited to N, P, O or S.

Preferably, the mRNA includes, but is not limited to, firefly luciferase mRNA, enhanced green fluorescent protein mRNA, or β galactosidase mRNA, and the like.

Preferably, said R is ₁ 、R ₂ 、R ₃ Each independently selected from any one of C6-C20 (for example, C8, C10, C12, C14, C15, C16, C17, C18, C19 or C20) straight-chain alkyl or C6-C20 (for example, C8, C10, C12, C14, C15, C16, C17, C18, C19 or C20) straight-chain alkenyl.

Preferably, said R is ₁ 、R ₂ 、R ₃ Each independently selected from any one of the following groups:

wherein the wavy line indicates the bond of the group.

Preferably, said R is ₄ ^- Is selected from Br ^- 、I ^- 、Cl ^- 、OH ^- Or NO ₃ ^- More preferably I ^- 。

Preferably, n is 1.

Preferably, the quaternary ammonium lipid-like molecule has any one of the following structures:

preferably, the quaternary ammonium lipid-like molecule is qtB-UC 18.

The molar ratio of N atoms in the quaternary ammonium salt lipid-like molecule to P atoms in mRNA (referred to as nitrogen-phosphorus ratio) is preferably (0.1 to 20):1, and may be, for example, 0.2:1, 0.3:1, 0.5:1, 0.6:1, 0.8:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1, and more preferably (1.5 to 15): 1.

Preferably, the lipid-like nanoparticle further comprises lipid molecules, and the structure of the lipid molecules is different from that of the quaternary ammonium salt lipid-like molecules.

Preferably, the molar ratio of the quaternary ammonium salt lipid-like molecules to the lipid molecules in the lipid-like nanoparticles is (0.1-10): 1, and for example, may be 0.2:1, 0.3:1, 0.5:1, 0.6:1, 0.8:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10: 1.

Preferably, the lipid molecule comprises any one of a non-cationic lipid, a cationic lipid or a polyethylene glycol modified lipid or a combination of at least two thereof.

Preferably, the non-cationic lipid comprises 1, 2-dicaprylyl-sn-glycero-3-phosphocholine (DEPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), Hydrogenated Soy Phosphatidylcholine (HSPC), 1-palmitoyl-2-oleoyl lecithin (POPC), 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC), 1, 2-myristic-sn-glycero-3-phosphorylethanolamine (DMPE), 1, 2-dioleyl-sn-glycero-3-phosphorylethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), dioleoyl phosphatidylglycerol (DOPG), 1, 2-palmitoyl phosphatidylglycerol (DPPG), 1, 2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), Cholesterol (Chol), 3 beta- [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol), sphingomyelin, ceramide, cephalin, cerebroside, diacylglycerol or sphingomyelin.

Preferably, the cationic lipid comprises N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxypropylamine (DODMA), 1, 2-dioleyloxy-3- (dimethylamino) acetoxypropane (DLin-DAC), 1, 2-dioleyl-3-dimethylaminopropane (DLin), 1-linoleoyl-2-linoleoxy-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-diolexy-3- (N-methylpiperazinyl) propane (DLin-MPZ), 3- (N, N-dioleylamino) -1, 2-propanediol (DOAP), 1, 2-dioleyloxy-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DMA), 1, 2-dilinolenyloxy-N, any one of N-dimethylaminopropane (DLinDMA) or 2, 2-dioleyl-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA) or a combination of at least two thereof.

Preferably, the polyethylene glycol (PEG) modified lipid comprises any one of PEG-phosphatidylethanolamine, PEG-phosphatidic acid, PEG-ceramide, PEG-dialkyl amine or PEG-diglyceride or a combination of at least two thereof; typically, any one or a combination of at least two of DMG-PEG, DLPE-PEG, DMPE-PEG, DPPC-PEG or DSPE-PEG is included.

Preferably, the weight average molecular weight Mw of the polyethylene glycol in the polyethylene glycol modified lipid is 1000 to 10000, such as 2000, 3000, 4000, 5000, 6000, 7000, 8000 or 9000.

Preferably, the mRNA delivery system has a hydrated particle size of 100 to 400nm, for example, 120nm, 140nm, 150nm, 160nm, 180nm, 200nm, 210nm, 230nm, 250nm, 270nm, 290nm, 300nm, 310nm, 330nm, 350nm, 370nm, or 390nm, and specific values therebetween, not to be limited in space and for the sake of brevity, the present invention is not exhaustive of the specific values included in the range.

In the mRNA delivery system provided by the present invention, the carrier includes lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules and optionally lipid molecules, which has the following advantages:

(1) good stability between batches

Lipid nanoparticles for in vivo delivery of mRNA disclosed in the prior art mainly consist of four or more components, with multiple components and complex formulation; the lipid-like nanoparticles can be formed by single components (quaternary ammonium salt lipid-like molecules) or double components (combination of quaternary ammonium salt lipid-like molecules and lipid molecules), have simple formula and simplified preparation process, and are beneficial to large-scale production and maintenance of quality stability among batches.

(2) High transfection efficiency

In the mRNA delivery system provided by the invention, through the formula optimization of the vector, the efficiency of in vitro delivery of mRNA can reach more than 5 times of that of a commercial transfection reagent lipofectamine 2K (Saimerfei).

(3) Efficient, highly specific lung-targeted delivery of mRNA

In the mRNA delivery system, efficient and high-specificity lung targeting mRNA delivery can be obtained by optimizing the carrier, namely adjusting the preparation ratio of single component (quaternary ammonium salt lipid-like molecules) or double component (combination of quaternary ammonium salt lipid-like molecules and lipid molecules) and mRNA.

In a second aspect, the present invention provides a method of preparing an mRNA delivery system according to the first aspect, the method comprising: mixing quaternary ammonium salt lipid-like molecules with an organic solvent to obtain an organic phase; mixing the organic phase with water to obtain a mixed phase; mixing the mixed phase with an mRNA solution to obtain the mRNA delivery system.

Preferably, the organic solvent is an organic solvent miscible with water, and more preferably an alcohol solvent.

Preferably, the organic solvent comprises methanol and/or ethanol.

Preferably, the concentration of the quaternary ammonium salt lipid-like molecules in the organic phase is 0.2-20 mg/mL, such as 0.5mg/mL, 0.8mg/mL, 1mg/mL, 3mg/mL, 5mg/mL, 7mg/mL, 9mg/mL, 10mg/mL, 11mg/mL, 13mg/mL, 15mg/mL, 17mg/mL or 19mg/mL, and specific values therebetween are not limited to the space and for the sake of brevity, and the invention does not list the specific values included in the range.

Preferably, the organic phase further comprises lipid molecules having a structure different from that of the quaternary ammonium salt lipid-like molecules.

The molar ratio of the lipid molecules to the quaternary ammonium salt lipid-like molecules is preferably 1: 0.1 to 10, and may be, for example, 1:0.2, 1:0.3, 1:0.5, 1:0.7, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1: 10.

The volume ratio of the organic phase to water is preferably 1 (0.25 to 19), and may be, for example, 1:0.3, 1:0.4, 1:0.5, 1:0.7, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:13, 1:15, 1:17, or 1:19, and more preferably 1 (1 to 10).

Preferably, the solvent of the mRNA solution is a buffer solution (aqueous solvent), and more preferably a PBS buffer solution.

Preferably, the concentration of mRNA in the mRNA solution is 5-500 ng/. mu.L, such as 8 ng/. mu.L, 10 ng/. mu.L, 15 ng/. mu.L, 20 ng/. mu.L, 25 ng/. mu.L, 30 ng/. mu.L, 40 ng/. mu.L, 50 ng/. mu.L, 100 ng/. mu.L, 150 ng/. mu.L, 200 ng/. mu.L, 250 ng/. mu.L, 300 ng/. mu.L, 350 ng/. mu.L, 400 ng/. mu.L or 450 ng/. mu.L, and the specific values therebetween are limited by space and for brevity, the invention is not exhaustive list of the specific values included in the range.

The volume ratio of the mixed phase to the mRNA solution is preferably 1 (0.25 to 19), and may be, for example, 1:0.3, 1:0.4, 1:0.5, 1:0.7, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:13, 1:15, 1:17, or 1:19, and more preferably 1 (1 to 10).

In the preparation method, the molar ratio of the N atom in the quaternary ammonium salt lipid-like molecule to the P atom in the mRNA (referred to as nitrogen-phosphorus ratio) in the system is preferably controlled to be (0.1 to 20):1, and may be, for example, 0.2:1, 0.3:1, 0.5:1, 0.6:1, 0.8:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1 or 19: 1.

Preferably, the preparation method specifically comprises the following steps: uniformly mixing quaternary ammonium salt lipid-like molecules, an organic solvent and optionally lipid molecules to obtain an organic phase; uniformly mixing the organic phase and water according to the volume ratio of 1 (0.25-19) and preferably 1 (1-10) to obtain a mixed phase; uniformly mixing mRNA with an aqueous solvent to obtain an mRNA solution; and (3) uniformly mixing the mixed phase with an mRNA solution in a volume ratio of 1 (0.25-19), preferably 1 (1-10) to obtain the mRNA delivery system.

In a third aspect, the present invention provides a use of an mRNA delivery system according to the first aspect in a lung targeting medicament.

The mRNA delivery system provided by the invention has high-efficiency and high-specificity lung targeting property, and is particularly suitable for being applied to lung targeting drugs/drugs for treating lung diseases.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising the mRNA delivery system of the first aspect.

Preferably, the administration mode of the pharmaceutical composition includes intramuscular injection, intradermal injection, intravenous injection or arterial injection, and more preferably intramuscular injection or intravenous injection.

Preferably, the pharmaceutical composition is a lung-targeted pharmaceutical composition.

Preferably, the pharmaceutical composition comprises a pharmaceutical composition for treating a pulmonary disease.

Preferably, the pharmaceutical composition further comprises an adjuvant.

Preferably, the adjuvant comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

Compared with the prior art, the invention has the following beneficial effects:

in the mRNA delivery system provided by the invention, the lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules are used as a carrier, so that the lung-targeted mRNA delivery with high efficiency and high specificity is obtained, the stability is excellent, the biocompatibility is good, the components are simple, the preparation process is simplified, the stability among batches is good, and a foundation is laid for the application of mRNA medicaments in the treatment of lung diseases.

Drawings

FIG. 1 is a graph showing the results of in vitro delivery efficiency of the mRNA delivery systems provided in examples 1 to 5 and 8 to 12;

FIG. 2 is a bioluminescence image of the whole mouse after administration of the mRNA delivery system provided in example 13;

FIG. 3 is a bioluminescence image of ex vivo organs of mice after administration of the mRNA delivery systems provided in examples 6, 7, 13, and 14;

FIG. 4 is a graph showing the results of total photon flux per gram of mouse organ after administration of the mRNA delivery systems provided in examples 6, 7, 13, and 14;

FIG. 5 is a graph of the results of the percentage of total photon flux per gram of organ in mice after administration of the mRNA delivery systems provided in examples 6, 7, 13, and 14;

FIG. 6 is a nucleic acid electropherogram of the mRNA delivery system provided in examples 1, 4, 6, 7, 10, 13, and 14;

FIG. 7 is a hydrated particle size histogram of the mRNA delivery system provided in examples 1, 4, 6, 7, 10, 13, 14;

FIG. 8 is a transmission electron micrograph of the mRNA delivery system provided in example 7;

FIG. 9 is a transmission electron micrograph of an mRNA delivery system provided in example 13;

FIG. 10 is a graph of stability in serum of the mRNA delivery systems provided in examples 6, 7, and 13.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

In a specific embodiment, the method for synthesizing the quaternary ammonium salt fatty-like molecule qtB-UC18 comprises the following steps:

(1) reacting mesitylene-trimethyl aldehyde with

Mixing according to the molar ratio of 1:3.6, dissolving in a mixed solvent of ethanol and dichloromethane, reacting at 35 ℃ for 24h, and adding NaBH ₄ Reducing at room temperature for 24h, and finally using CHCl ₃ Extracting for 3 times; purifying the extract product with medium pressure preparative chromatography (lambda) ₁ ＝216nm、λ ₂ 254nm), with CH ₂ Cl ₂ And methanol is taken as a mobile phase for purification, and the intermediate tB-UC18 is obtained by decompression, concentration and drying.

(2) Dissolving the obtained tB-UC18 in ethanol, adding 6 times of iodomethane by molar weight by taking potassium carbonate as an acid-binding agent, and stirring in an oil bath at 39 ℃ for 48 hours. Performing rotary evaporation under reduced pressure, adding dichloromethane, washing with water for three times, and performing rotary evaporation on the organic solvent again to obtain a product qtB-UC 18.

qtB-UC18 mass-to-charge ratio theoretical value is 334.3468, and mass spectrum detection value is 334.3612;

¹ H-NMR(400MHz，CDCl ₃ )：δ＝8.24(s,3H),5.34(t,J＝5.8Hz,6H),4.96(s,6H),3.65(s,18H),3.37(d,J＝18.1Hz,6H),2.01(dd,J＝12.7,6.7Hz,12H),1.82(s,6H),1.47–1.14(m,66H),0.88(t,J＝6.7Hz,9H)。

example 1

An mRNA delivery system comprises a carrier and firefly luciferase mRNA (provided by Shanghai Wei atlas biotechnology limited) loaded on the carrier, wherein the carrier is lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules qtB-UC 18; qtB-UC18, wherein the molar ratio of N atoms in the mRNA to P atoms in the mRNA (hereinafter referred to as nitrogen-phosphorus ratio) is 1.5: 1; the preparation method comprises the following steps:

(1) dissolving mRNA into a PBS buffer solution with the pH value of 7.4 to obtain an mRNA solution with the concentration of 28 ng/. mu.L;

(2) dissolving quaternary ammonium salt lipid-like molecules qtB-UC18 in ethanol to obtain an organic phase with the concentration of 1.5 mg/mL; uniformly mixing the organic phase and water in a volume ratio of 1:9 to obtain a mixed phase;

(3) and (3) uniformly mixing the mRNA solution obtained in the step (1) and the mixed phase obtained in the step (2) in a volume ratio of 3:7 to ensure that the nitrogen-phosphorus ratio of qtB-UC18 to mRNA is 1.5:1, thus obtaining the mRNA delivery system.

Examples 2 to 5

An mRNA delivery system differing from example 1 only in the concentrations of qtB-UC18, such that the nitrogen to phosphorus ratios of qtB-UC18 to mRNA were different, 2:1, 3:1, 4.5:1, 6: 1; other components, the using amount and the preparation method are the same as those of the embodiment 1; specific nitrogen phosphorus ratios are shown in table 1.

Example 6

An mRNA delivery system comprises a carrier and firefly luciferase mRNA loaded on the carrier, wherein the carrier is lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules qtB-UC 18; qtB-UC18, wherein the molar ratio of N atoms to P atoms in mRNA (hereinafter referred to as nitrogen-phosphorus ratio) is 9: 1; the preparation method comprises the following steps:

(1) dissolving mRNA into a PBS buffer solution with the pH value of 7.4 to obtain an mRNA solution with the concentration of 70 ng/. mu.L;

(2) dissolving quaternary ammonium salt lipid-like molecules qtB-UC18 in ethanol to obtain an organic phase with the concentration of 7.5 mg/mL; uniformly mixing the organic phase and water in a volume ratio of 3:7 to obtain a mixed phase;

(3) and (3) uniformly mixing the mRNA solution obtained in the step (1) and the mixed phase obtained in the step (2) in a volume ratio of 3:7 to ensure that the nitrogen-phosphorus ratio of qtB-UC18 to mRNA is 9:1, thus obtaining the mRNA delivery system. Specific nitrogen phosphorus ratios are shown in table 1.

Example 7

An mRNA delivery system comprises a carrier and firefly luciferase mRNA loaded on the carrier, wherein the carrier is lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules qtB-UC 18; qtB-UC18, wherein the molar ratio of N atoms to P atoms in mRNA (hereinafter referred to as nitrogen-phosphorus ratio) is 15: 1; the preparation method comprises the following steps:

(2) dissolving quaternary ammonium salt lipid-like molecules qtB-UC18 in ethanol to obtain an organic phase with the concentration of 8.3 mg/mL; uniformly mixing the organic phase and water in a volume ratio of 4.5:5.5 to obtain a mixed phase;

(3) and (3) uniformly mixing the mRNA solution obtained in the step (1) and the mixed phase obtained in the step (2) in a volume ratio of 3:7, so that the nitrogen-phosphorus ratio of qtB-UC18 to mRNA is 15:1, and thus obtaining the mRNA delivery system. Specific nitrogen phosphorus ratios are shown in table 1.

TABLE 1

	Nitrogen to phosphorus ratio
		Example 1	1.5:1
Example 2	2:1
		Example 3	3:1
Example 4	4.5:1
		Example 5	6:1
Example 6	9:1
		Example 7	15:1

Example 8

An mRNA delivery system comprises a carrier and firefly luciferase mRNA loaded on the carrier, wherein the carrier is lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules qtB-UC18 and lipid molecules (DOPE, 1, 2-dioleyl-sn-glycerol-3-phosphorylethanolamine); qtB-UC18 and mRNA have nitrogen-phosphorus ratio of 1.5:1, and qtB-UC18 and DOPE have molar ratio of 4: 1; the preparation method comprises the following steps:

(2) qtB-UC18 and DOPE are dissolved in ethanol according to the molar ratio of 4:1 to obtain an organic phase with the concentration of qtB-UC18 of 3mg/mL and the concentration of DOPE of 0.4 mg/mL; uniformly mixing the organic phase and water in a volume ratio of 1:9 to obtain a mixed phase;

(3) and (3) uniformly mixing the mRNA solution obtained in the step (1) and the mixed phase obtained in the step (2) in a volume ratio of 3:7 to obtain an mRNA delivery system, wherein the nitrogen-phosphorus ratio of qtB-UC18 to mRNA is 1.5:1, and the molar ratio of qtB-UC18 to DOPE is 4: 1.

Examples 9 to 12

An mRNA delivery system differing from example 8 only in the molar ratio of qtB-UC18 to the lipid molecule DOPE; other components, the using amount and the preparation method are the same as those of the embodiment 8; the specific formulation is shown in table 2.

Example 13

An mRNA delivery system comprises a carrier and firefly luciferase mRNA loaded on the carrier, wherein the carrier is lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules qtB-UC 18; qtB-UC18 and mRNA have nitrogen-phosphorus ratio of 6:1, and qtB-UC18 and DOPE have molar ratio of 1: 1; the preparation method comprises the following steps:

(2) qtB-UC18 and DOPE are dissolved in ethanol according to the molar ratio of 1:1 to obtain an organic phase with the concentration of qtB-UC18 of 9.9mg/mL and the concentration of DOPE of 5.3 mg/mL; uniformly mixing the organic phase and water in a volume ratio of 3:7 to obtain a mixed phase;

(3) and (3) uniformly mixing the mRNA solution obtained in the step (1) and the mixed phase obtained in the step (2) in a volume ratio of 3:7 to ensure that the nitrogen-phosphorus ratio of qtB-UC18 to mRNA is 6:1, and the molar ratio of qtB-UC18 to DOPE is 1:1 to obtain the mRNA delivery system. The specific formulation is shown in table 2.

Example 14

An mRNA delivery system comprises a carrier and firefly luciferase mRNA loaded on the carrier, wherein the carrier is lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules qtB-UC 18; the nitrogen-phosphorus ratio of qtB-UC18 to mRNA is 9:1, and the molar ratio of qtB-UC18 to DOPE is 1: 1; the preparation method comprises the following steps:

(2) qtB-UC18 and DOPE are dissolved in ethanol according to the molar ratio of 1:1 to obtain an organic phase with the concentration of qtB-UC18 of 9.9mg/mL and the concentration of DOPE of 5.3 mg/mL; uniformly mixing the organic phase and water in a volume ratio of 4.5:5.5 to obtain a mixed phase;

(3) and (3) uniformly mixing the mRNA solution obtained in the step (1) and the mixed phase obtained in the step (2) in a volume ratio of 3:7, so that the nitrogen-phosphorus ratio of qtB-UC18 to mRNA is 9:1, and the molar ratio of qtB-UC18 to DOPE is 1:1, thereby obtaining the mRNA delivery system. The specific formulation is shown in table 2.

TABLE 2

	qtB-UC18 to DOPE molar ratio	Nitrogen to phosphorus ratio
			Example 8	4:1	1.5:1
Example 9	2:1	1.5:1
			Example 10	1:1	1.5:1
Example 11	1:2	1.5:1
			Example 12	1:4	1.5:1
Example 13	1:1	6:1
			Example 14	1:1	9:1

The performance test and analysis of the mRNA delivery systems provided in examples 1 to 14 above were performed as follows:

firstly, optimizing a preparation formula at a cellular level, wherein the method comprises the following steps:

293T cells are paved in a 96-well plate, each well contains 90 mu L of whole cell culture solution, after 24h, 10 mu L of mRNA delivery system to be detected (examples 1-5 and 8-12) is added into the well and placed in a cell incubator for culture, and after 24h, the expression of protein is detected; lipofectamine 2K (abbreviated as Lipo 2K) from semer fly was used as a reference.

The optimization of the formulation is carried out by two schemes: (1) optimizing the nitrogen-phosphorus ratio of the quaternary ammonium salt lipid-like molecules to mRNA for a single-component mRNA delivery system; (2) for a two-component mRNA delivery system, the nitrogen-to-phosphorus ratio of the quaternary ammonium lipidlike molecules to the mRNA is fixed first, and then the molar ratio of the quaternary ammonium lipidlike molecules to the lipid molecules is optimized.

The results of the in vitro delivery efficiency test of the mRNA delivery system obtained by the above-described protocol are shown in fig. 1, and the relative light intensity (%) on the ordinate is in terms of the intensity of Lipo 2K (i.e., Lipo 2K is 100%). Corresponding to the optimization scheme of the first formulation (examples 1-5), the in vitro delivery efficiency of example 4 was the highest, 516%; corresponding to the optimization of the second formulation (examples 8-12), the in vitro delivery efficiency of example 10 was the highest at 484%.

Second, in vivo targeting evaluation of mRNA delivery System

C57BL/6J mice are used as experimental objects, and the mRNA delivery system provided by the invention is injected into the mice by means of tail vein injection administration (for simplicity, the in-vivo experimental results of the mice of examples 6, 7, 13 and 14 are provided exemplarily), and each mouse is administered with 0.5mg of mRNA/kg of tail vein; 4h after administration, the fluorogenic enzyme substrate is intraperitoneally administered at a dose of 150mg/kg, and 10min later, the mice are subjected to whole body imaging by a small animal imager (Perkinelmer); then, main organs (heart, liver, spleen, lung, kidney, etc.) are taken out in sequence for in vitro imaging, and the protein expression condition of the firefly luciferase is detected. The mRNA lipid-like nano-delivery system tB-UC18 LLNs with the nitrogen-phosphorus ratio of tB-UC18 to mRNA being 1.5:1 and the molar ratio of tB-UC18 to DOPE being 1:1, which are reported previously, are used as a control group (tB-UC 18 and DOPE are dissolved in ethanol in the molar ratio of 1:1 to form an organic phase, and the organic phase and a firefly luciferase mRNA solution are directly and uniformly mixed in the volume ratio of 1:9 to obtain the mRNA lipid-like nano-delivery system control group).

Illustratively, the bioluminescence profile of the global imaging of mice 4h after tail vein injection of the mRNA delivery system provided in example 13 or the control group is shown in fig. 2; bioluminescence profile of ex vivo imaging of mouse organs 4h after mice were injected via tail vein with mRNA delivery system provided in examples 6, 7, 13, 14 or control group is shown in figure 3; the Total photon flux values per gram of mouse tissue (in Total flux/g) obtained by quantitative analysis of photon flux (Total flux) of the bioluminescence of fig. 3 by perkin elmer live imaging Software (Living Image Software) are shown in fig. 4; the percentage results calculated from the total photon flux values of figure 3 per gram of mouse tissue are shown in figure 5; as can be seen from fig. 2 to 5, the single-component (quaternary ammonium salt lipid-like molecule) mRNA delivery systems (examples 6 and 7) and the two-component (combination of quaternary ammonium salt lipid-like molecule and lipid molecule) mRNA delivery systems (examples 13 and 14) achieve high-efficiency and high-specificity expression of 77% to 95% of mRNA in the lung; the mRNA lipid-like nano-delivery system tB-UC18 LLNs used as the control group cannot realize lung targeting. The above results indicate that other types of mRNA lipid-like nano delivery systems prepared by other methods cannot achieve lung targeting, while the mRNA delivery system obtained by the specific carrier and preparation method using the specific formulation provided by the present invention achieves highly specific lung targeting.

Third, evaluation of load Performance of mRNA delivery System

The loading performance of the mRNA delivery system provided by the present invention on mRNA was evaluated by nucleic acid electrophoresis, and as shown in fig. 6, for example, fig. 1, 4, 6, 7, 10, 13, and 14, the electrophoretic bands of the mRNA delivery system migrated relative to the electrophoretic bands of free mRNA, which indicates that the quaternary ammonium lipid-like molecules can successfully load mRNA either alone or in combination with lipid molecules.

IV, hydrated particle size of mRNA delivery system

The mRNA delivery system provided by the present invention is characterized by hydrated particle size using DLS (dynamic light scattering), and exemplarily, hydrated particle sizes of the mRNA delivery systems provided by examples 1, 4, 6, 7, 10, 13, and 14 are counted, and an obtained hydrated particle size statistical graph is shown in fig. 7, which shows that the hydrated particle size range of the mRNA delivery system is 100 to 400 nm.

Fifth, morphological characterization of mRNA delivery systems

The morphology of the mRNA delivery System provided by the present invention is characterized by using a Hitachi TEM System transmission electron microscope, and exemplarily, a transmission electron microscope image of the mRNA delivery System provided in example 7 is shown in fig. 8, and the result shows that the mRNA delivery System has a regular spherical morphology, and the particle size is about 120 nm; the transmission electron micrograph of the mRNA delivery system provided in example 13 is shown in fig. 9, and the result shows that the mRNA delivery system has a regular spherical morphology and a particle size of about 300 nm.

Sixthly, stability evaluation of mRNA delivery System

After the mRNA delivery system provided by the invention is incubated in 10% serum at room temperature, the stability of the mRNA delivery system is monitored by detecting the absorbance change (660nm) of the serum at different time points. Exemplarily, serum stability test patterns of the mRNA delivery systems provided in examples 6, 7, and 13 are shown in fig. 10, and after the mRNA delivery systems are incubated in 10% serum at different times (6h, 24h, 48h, and 72h), the absorbance value at 660nm is not significantly changed relative to 0h, which indicates that the mRNA delivery systems are not significantly aggregated in the measurement range, and have good stability.

The applicant states that the present invention is illustrated by the above examples of the mRNA delivery system of the present invention and the preparation method and application thereof, but the present invention is not limited to the above process steps, i.e. it does not mean that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modifications to the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific forms, etc., are within the scope and disclosure of the present invention.

Claims

1. An mRNA delivery system, characterized in that the mRNA delivery system comprises a carrier and mRNA loaded on the carrier; the carrier comprises lipid-like nanoparticles containing quaternary ammonium salt lipid-like molecules; the quaternary ammonium salt lipid-like molecule has the following structure:

the molar ratio of N atoms in the quaternary ammonium salt lipid-like molecules to P atoms in mRNA is (1.5-15): 1, and the hydrated particle size of the mRNA delivery system is 100-400 nm;

2. The mRNA delivery system of claim 1, wherein the lipid-like nanoparticles further comprise lipid molecules having a structure that is different from the structure of the quaternary ammonium salt lipid-like molecules.

3. The mRNA delivery system according to claim 2, wherein the molar ratio of quaternary ammonium salt lipid-like molecules to lipid molecules in the lipid-like nanoparticles is (0.1-10): 1.

4. The mRNA delivery system of claim 2, wherein the lipid molecule is any one of or a combination of at least two of a non-cationic lipid, a cationic lipid, or a polyethylene glycol modified lipid.

5. The mRNA delivery system of claim 4, wherein the non-cationic lipid is 1, 2-dicaprylyl-sn-glycero-3-phosphocholine, 1, 2-dimyristoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, hydrogenated soy phosphatidylcholine, 1-palmitoyl-2-oleoyl lecithin, 1-stearoyl-2-oleoyl phosphatidylcholine, 1, 2-myristic-sn-glycero-3-phosphorylethanolamine, o, 1, 2-dioleyl-sn-glycero-3-phosphoethanolamine, 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, distearoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidyethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, 1, 2-dimyristoyl-sn-glycero-3-phosphoglycerol, dioleoylphosphatidylglycerol, 1, 2-palmitoylphosphatidylglycerol, 1, 2-distearoyl-sn-glycero-3-phosphoglycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, cholesterol, 3 β - [ N- (N', n' -dimethylaminoethyl) carbamoyl ] cholesterol, sphingomyelin, ceramide, cephalin, cerebroside, diacylglycerol, or a combination of at least two thereof.

6. The mRNA delivery system of claim 4, wherein the cationic lipid is N, N-dioleyl-N, N-dimethylammonium chloride, N, N-distearyl-N, N-dimethylammonium bromide, N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride, N, N-dimethyl-2, 3-dioleyloxypropylamine, 1, 2-dioleyloxy-3- (dimethylamino) acetoxypropane, 1, 2-dioleyl-3-dimethylaminopropane, 1-linoleoyl-2-linoleoxy-3-dimethylaminopropane, N, N-dioleyl-2-linoleoxy-3-dimethylaminopropane, N, N-trimethylammoniumchloride, N, N-dioleyloxy) propyl-N, N, N, N-dioleyloxy-3-dimethylaminopropane, Any one or a combination of at least two of 1, 2-dioleyloxy-3- (N-methylpiperazinyl) propane, 3- (N, N-dioleylamino) -1, 2-propanediol, 1, 2-dioleyloxy-3- (2-N, N-dimethylamino) ethoxypropane, 1, 2-dilinolyloxy-N, N-dimethylaminopropane or 2, 2-dioleyl-4-dimethylaminomethyl- [1,3] -dioxolane.

7. The mRNA delivery system of claim 4, wherein the polyethylene glycol-modified lipid is any one of PEG-phosphatidylethanolamine, PEG-phosphatidic acid, PEG-ceramide, PEG-dialkylamine, or PEG-diglyceride, or a combination of at least two thereof.

8. A method of making an mRNA delivery system according to any one of claims 1 to 7, wherein the method comprises: mixing quaternary ammonium salt lipid-like molecules with an organic solvent to obtain an organic phase; mixing the organic phase with water to obtain a mixed phase; and mixing the mixed phase with an mRNA solution to obtain the mRNA delivery system.

9. The method according to claim 8, wherein the organic solvent is an alcohol solvent.

10. The method according to claim 8, wherein the organic solvent is methanol and/or ethanol.

11. The preparation method according to claim 8, wherein the concentration of the quaternary ammonium salt lipid-like molecules in the organic phase is 0.2-20 mg/mL.

12. The method of claim 8, wherein the organic phase further comprises lipid molecules having a structure different from that of the quaternary ammonium salt lipid-like molecules.

13. The method according to claim 12, wherein the molar ratio of the lipid molecules to the quaternary ammonium salt lipid-like molecules is 1 (0.1 to 10).

14. The method according to claim 8, wherein the volume ratio of the organic phase to water is 1 (0.25-19).

15. The method according to claim 8, wherein the volume ratio of the organic phase to water is 1 (1-10).

16. The method according to claim 8, wherein the solvent for the mRNA solution is a buffer solution.

17. The method of claim 8, wherein the mRNA solution is in PBS buffer.

18. The method according to claim 8, wherein the concentration of mRNA in the mRNA solution is 5 to 500ng/μ L.

19. The method according to claim 8, wherein the volume ratio of the mixed phase to the mRNA solution is 1 (0.25 to 19).

20. The method according to claim 8, wherein the volume ratio of the mixed phase to the mRNA solution is 1 (1-10).

21. Use of an mRNA delivery system according to any one of claims 1 to 7 for the preparation of a lung-targeted medicament.

22. A pharmaceutical composition comprising the mRNA delivery system of any one of claims 1 to 7.

23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is administered intramuscularly, intradermally, intravenously or intraarterially.

24. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is administered intramuscularly or intravenously.

25. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is a pharmaceutical composition for treating a pulmonary disease.

26. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition further comprises an adjuvant.

27. The pharmaceutical composition of claim 26, wherein the adjuvant is any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.