Intracellular Delivery System for mRNA Nucleic Acid Drugs, Preparation Method and Application Thereof

Abstract

A delivery system for mRNA nucleic acid drugs, a preparation method and an application thereof are provided. The delivery system includes lipid nanoparticles for loading one or more kinds of mRNA molecules, wherein the lipid nanoparticles are prepared from raw materials including an ionizable cationic lipid, a phospholipid auxiliary lipid, cholesterol, and a polyethylene glycol-derivatized phospholipid. In the mRNA nucleic acid drug targeted intracellular delivery system based on the non-viral carrier of the present invention, the mRNA is concentrated and loaded by the electrostatic interaction between the ionizable cationic lipid and the mRNA. Phospholipid auxiliary lipid component-mediated pH sensitivity and late endosomal escape enable mRNA nucleic acid drugs to be efficiently delivered to target cells and then released into the cytoplasm of the target cells for exerting a pharmacodynamic effect.

Claims

1. A delivery system for mRNA nucleic acid drugs, comprising lipid nanoparticles for loading one or more kinds of mRNA molecules, wherein the lipid nanoparticles are prepared from raw materials, and the raw materials comprise an ionizable cationic lipid, a phospholipid auxiliary lipid, cholesterol, and a polyethylene glycol-derivatized phospholipid.

2. The delivery system according to claim 1, wherein the ionizable cationic lipid contains a monovalent cationic amino group or a multivalent cationic amino group, and the ionizable cationic lipid is at least one selected from the group consisting of N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-3-trimethyl ammonium-propane (chloride salt) (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 3-(dimethylamino)propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa-12,15-dienoate (DMAP-BLP), 3B—[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol.HCl), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (Dlin-KC2-DMA), and/or, the phospholipid auxiliary lipid is at least one selected from the group consisting of 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOP S), 1,2-dimyristoyl-sn-glycero-3-P (DMPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and/or, the polyethylene glycol-derivatized phospholipid is at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG 2000), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000), and/or, the mRNA molecules are selected from intact mRNA molecules expressing functional proteins, therapeutic monoclonal antibodies, B cell epitopes, T cell epitopes, or tumor neoantigen peptides.

3. The delivery system according to claim 1, wherein a molar ratio of the ionizable cationic lipid, the phospholipid auxiliary lipid, the cholesterol, and the polyethylene glycol-derivatized phospholipid is (5-60):(5-35):(25-70):(0.2-15).

4. The delivery system according to claim 1, wherein an average particle size of the lipid nanoparticles is 50-100 nm; and/or, under a neutral environmental condition, a Zeta potential of mRNA/lipid nanoparticles ranges from +30 mV to +35 mV.

5. A method for preparing the delivery system for mRNA nucleic acid drugs according to claim 1, comprising the following steps: S1, completely dissolving the raw materials in a first organic solvent to obtain a mixture for mixing, and then removing the first organic solvent from the mixture by a rotary evaporation to obtain a thin lipid membrane, and removing a residual first organic solvent from the mixture by a vacuum drying to obtain a dried thin lipid membrane; S2, dissolving the dried thin lipid membrane in a second organic solvent to obtain a liquid; S3, mixing an mRNA solution with the liquid to obtain an mRNA/lipid nanoparticle suspension solution; and S4, purifying and concentrating the mRNA/lipid nanoparticle suspension solution to obtain mRNA/lipid nanoparticles for preservation; wherein the first organic solvent is chloroform; the second organic solvent is anhydrous ethanol; and in step S3, a mass ratio of mRNA molecules in the mRNA solution to the ionizable cationic lipid is 1:(10-20).

6. The method according to claim 5, wherein in step S1, the rotary evaporation is performed at a gauge pressure of 0.06 Mpa and 30-35° C., until a uniform thickness thin lipid membrane is formed at a bottom of a round bottom flask, and then the vacuum drying is performed at a gauge pressure of −0.1 Mpa and 25-30° C. for 4-6 h.

7. The method according to claim 5, wherein in step S3, the mRNA solution comprises a buffer for diluting an mRNA storage solution, and the buffer is at least one selected from the group consisting of a sodium citrate buffer having a concentration of 50 mM and a pH of 4.0, a sodium citrate buffer having a concentration of 10 mM and a pH of 3.0, a sodium citrate buffer having a concentration of 10 mM and a pH of 4.0, and a sodium acetate buffer having a concentration of 50 mM and a pH of 5.0.

8. The method according to claim 5, wherein in step S3, a flow rate ratio of the mRNA solution to the liquid and a total flow velocity of a mixing pipeline are controlled by a microfluidic method; and/or, in step S3, the flow rate ratio of the mRNA solution to the liquid is 1:(1-5); and/or in step S3, the total flow rate of the mixing pipeline of the mRNA solution and the liquid is 1 ml/min-12 ml/min.

9. The method according to claim 5, wherein in step S4, the mRNA/lipid nanoparticles are purified by a dialysis or a tangential flow filtration; an interception pore size of a dialysis membrane for the dialysis is 10 kd; a process of the dialysis for the mRNA/lipid nanoparticles comprises: dialyzing twice in a phosphate buffered saline (PBS) having a pH of 7.4 and a volume 200 times greater than or equal to a volume of the mRNA/lipid nanoparticles, a first dialysis is performed at room temperature (25° C.) for 2-4 h, and a second dialysis is performed at a low temperature of 4° C. for 12-18 h, with a total duration of the first dialysis and the second dialysis not less than 18 h; and/or in step S4, the mRNA/lipid nanoparticles are concentrated by a centrifugal ultrafiltration; an interception pore size of an ultrafiltration tube is 3 kd; the mRNA/lipid nanoparticles are concentrated by centrifuging with a fixed-angle rotor having an angle of 30-50 degrees and a weight of 14000 g at room temperature of 25° C. for 25-35 min; and/or in step S4, the mRNA/lipid nanoparticles are filtered by a 0.22 μm filter membrane for 5 times and a 0.1 μm filter membrane for 3 times, and then sub-packaged for preservation at −80° C.

10. A method of preparing a drug delivery system, comprising applying the delivery system according to any claim 1.

11. The delivery system according to claim 2, wherein a molar ratio of the ionizable cationic lipid, the phospholipid auxiliary lipid, the cholesterol, and the polyethylene glycol-derivatized phospholipid is (5-60):(5-35):(25-70):(0.2-15).

12. The delivery system according to claim 2, wherein an average particle size of the lipid nanoparticles is 50-100 nm; and/or, under a neutral environmental condition, a Zeta potential of mRNA/lipid nanoparticles ranges from +30 mV to +35 mV.

13. The delivery system according to claim 3, wherein an average particle size of the lipid nanoparticles is 50-100 nm; and/or, under a neutral environmental condition, a Zeta potential of mRNA/lipid nanoparticles ranges from +30 mV to +35 mV.

14. The method according to claim 5, wherein the ionizable cationic lipid contains a monovalent cationic amino group or a multivalent cationic amino group, and the ionizable cationic lipid is at least one selected from the group consisting of N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butyl carboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-3-trimethyl ammonium-propane (chloride salt) (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 3-(dimethylamino)propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa-12,15-dienoate (DMAP-BLP), 3B—[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol.HCl), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (Dlin-KC2-DMA), and/or, the phospholipid auxiliary lipid is at least one selected from the group consisting of 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOP S), 1,2-dimyristoyl-sn-glycero-3-P (DMPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and/or, the polyethylene glycol-derivatized phospholipid is at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG 2000), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000), and/or, the mRNA molecules are selected from intact mRNA molecules expressing functional proteins, therapeutic monoclonal antibodies, B cell epitopes, T cell epitopes, or tumor neoantigen peptides.

15. The method according to claim 5, wherein a molar ratio of the ionizable cationic lipid, the phospholipid auxiliary lipid, the cholesterol, and the polyethylene glycol-derivatized phospholipid is (5-60):(5-35):(25-70):(0.2-15).

16. The method according to claim 5, wherein an average particle size of the lipid nanoparticles is 50-100 nm; and/or, under a neutral environmental condition, a Zeta potential of mRNA/lipid nanoparticles ranges from +30 mV to +35 mV.

17. The method according to claim 6, wherein in step S3, the mRNA solution comprises a buffer for diluting an mRNA storage solution, and the buffer is at least one selected from the group consisting of a sodium citrate buffer having a concentration of 50 mM and a pH of 4.0, a sodium citrate buffer having a concentration of 10 mM and a pH of 3.0, a sodium citrate buffer having a concentration of 10 mM and a pH of 4.0, and a sodium acetate buffer having a concentration of 50 mM and a pH of 5.0.

18. The method according to claim 6, wherein in step S3, a flow rate ratio of the mRNA solution to the liquid and a total flow velocity of a mixing pipeline are controlled by a microfluidic method; and/or, in step S3, the flow rate ratio of the mRNA solution to the liquid is 1:(1-5); and/or in step S3, the total flow rate of the mixing pipeline of the mRNA solution and the liquid is 1 ml/min-12 ml/min.

19. The method according to claim 7, wherein in step S3, a flow rate ratio of the mRNA solution to the liquid and a total flow velocity of a mixing pipeline are controlled by a microfluidic method; and/or, in step S3, the flow rate ratio of the mRNA solution to the liquid is 1:(1-5); and/or in step S3, the total flow rate of the mixing pipeline of the mRNA solution and the liquid is 1 ml/min-12 ml/min.

20. The method according to claim 6, wherein in step S4, the mRNA/lipid nanoparticles are purified by a dialysis or a tangential flow filtration; an interception pore size of a dialysis membrane for the dialysis is 10 kd; a process of the dialysis for the mRNA/lipid nanoparticles comprises: dialyzing twice in a phosphate buffered saline (PBS) having a pH of 7.4 and a volume 200 times greater than or equal to a volume of the mRNA/lipid nanoparticles, a first dialysis is performed at room temperature (25° C.) for 2-4 h, and a second dialysis is performed at a low temperature of 4° C. for 12-18 h, with a total duration of the first dialysis and the second dialysis not less than 18 h; and/or in step S4, the mRNA/lipid nanoparticles are concentrated by a centrifugal ultrafiltration; an interception pore size of an ultrafiltration tube is 3 kd; the mRNA/lipid nanoparticles are concentrated by centrifuging with a fixed-angle rotor having an angle of 30-50 degrees and a weight of 14000 g at room temperature of 25° C. for 25-35 min; and/or in step S4, the mRNA/lipid nanoparticles are filtered by a 0.22 μm filter membrane for 5 times and a 0.1 μm filter membrane for 3 times, and then sub-packaged for preservation at −80° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 is a schematic diagram showing the principle of an intracellular delivery system for mRNA nucleic acid drugs of the present invention;

[0043] FIG. 2 shows a potential change of the mRNA/lipid nanoparticles in some embodiments of the present invention at different pH values;

[0044] FIG. 3 shows a qualitative analysis of encapsulation efficiency of mRNA/lipid nanoparticles in some embodiments of the present invention; and

[0045] FIG. 4A shows intracellular transfection efficiency of mRNA nucleic acid drugs according to some embodiments of the present invention by fluorescence microscope.

[0046] FIG. 4B shows intracellular transfection efficiency of mRNA nucleic acid drugs according to some embodiments of the present invention by flow cytometry.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] The present invention is illustrated with the following specific embodiments. Those skilled in the art can easily understand the other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can further be implemented or applied through different specific embodiments, and the details in this specification can also be modified or changed without deviating from the spirit of the present invention based on different viewpoints and applications.

[0048] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; furthermore, the terms used in the embodiments of the present invention are intended to describe the specific embodiments, rather than limit the protection scope of the present invention.

[0049] When a numerical range is presented in specific embodiments, it should be understood that unless otherwise stated in the present invention, two endpoints of each numerical range and any one value between the two endpoints can be selected for the present invention. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those skilled in the technical field generally understand. In addition to the specific methods, devices and materials used in the embodiments, according to existing techniques known by those skilled in the art and the disclosure of the present invention, the present invention can further be realized by using any method, device and material of the existing techniques similar to or equivalent to the methods, devices and materials described in the embodiments of the present invention.

[0050] The materials, reagents, etc., used in the following embodiments, unless otherwise specified, are commercially available.

Definition

[0051] The terms “prevention” and the like mean to exempt or significantly reduce the incidence of a disease by vaccinating healthy and normal people before the disease occurs.

[0052] The terms “treatment” and the like mean to alleviate or slow at least one symptom associated with a condition, or to slow or reverse the development of the condition, such as slowing or reversing the development of liver cancer.

[0053] The terms “endosome” and the like refer to a kind of membrane-wrapped vesicle structure, which can be divided into an early endosome and a late endosome. The early endosome is usually located on the outside of the cytoplasm. The late endosome is usually located on the inside of the cytoplasm, close to the cell nucleus. The late endosome contains a variety of hydrolases in the acidic internal environment.

[0054] The terms “protonation” and the like refer to a process by which an atom, molecule, or ion acquires proton (H.sup.+). It can be understood simply as a combination of a lone pair electron and a proton, that is, to combine one proton. Generally, this substance has lone pair electrons, and each of the lone pair electrons can bind one proton through a coordination bond.

[0055] The terms “B cell epitope” and the like refer to a sequence fragment or spatial conformation that can be specifically recognized and bound by B cell receptors (BCRs) or antibodies in antigenic molecules such as proteins, sugars, lipids, etc.

[0056] The terms “T cell epitope” and the like refer to a short peptide sequence presented by a major histocompatibility complex (MHC) molecule to a T cell receptor (TCR) after a protein antigen is processed by an antigen presenting cell (APC), which is generally a linear epitope.

[0057] The terms “tumor neoantigen” and the like refer to an antigen peptide fragment that exists on the surface of tumor cells in the form of MHC-peptide complex, which is produced by somatic gene mutation of tumor cells and closely bound to a major histocompatibility complex (MHC) molecule, and can be specifically recognized by the T cell receptor (TCR), thus activating the immune response of T cells.

[0058] As used in the present disclosure, the “mRNA/lipid nanoparticles” includes pharmaceutically effective amounts of mRNA, and pharmaceutically acceptable mRNA drug delivery carriers which can be used on a large scale in clinical applications.

[0059] As used in the present disclosure, the “transfected cell” is the cell in which an mRNA molecule has been introduced and a corresponding protein can be translated and expressed by the mRNA molecule.

[0060] In the following embodiments, the mRNA nucleic acid drug molecules are obtained by in vitro transcription. The cholesterol for regulating membrane fluidity is selected from a pharmaceutical-grade cholesterol derived from wool with a purity of more than 98%. The microfluidic control is mainly realized by NanoAssemblr®Benchtop nanoparticle synthesis system and the software PRECISION Nanosystems thereof.

EMBODIMENTS

[0061] The principle of the intracellular delivery system for mRNA nucleic acid drugs prepared in the present invention is shown in FIG. 1. The delivery system for mRNA nucleic acid drugs is a complex formed by lipid nanoparticles encapsulating and loading the mRNA nucleic acid drugs. The lipid nanoparticles are composed of an ionizable cationic lipid, a phospholipid auxiliary lipid, cholesterol, and a polyethylene glycol-derivatized phospholipid in a certain ratio. Among them, the ionizable cationic lipid contains monovalent or multivalent cationic amino groups. These cationic amino groups can concentrate and load the mRNA molecules through electrostatic interaction with the negatively charged mRNA molecules. The phospholipid auxiliary lipid is sensitive to environmental pH changes, which help the lipid nanoparticles escape from late endosomes (The mRNA nucleic acid drugs are effectively delivered into the target cells and released in the acidic environment of the late endosomes having a specific pH in the target cells, so that the nucleic acid drugs are released into the cytoplasm for translation and expression into proteins, thereby exerting its function). Moreover, the phospholipid auxiliary lipid can also increase the membrane stability and mRNA transfection efficiency. The cholesterol can regulate the membrane fluidity. The polyethylene glycol-derivatized phospholipid (PEG-lipid) can increase the hydrophilicity of the surface of the lipid nanoparticles and reduce the non-specific adsorption of the lipid nanoparticles to proteins in serum or tissue fluid, thus reducing the immunogenicity of the lipid nanoparticles.

[0062] It is accepted that effective gene delivery requires a large molar charge ratio (cationic lipid/nucleic acid), but with the increase of the ionizable cationic lipid content, the damage to the cell membrane will increase, and the cytotoxicity of the prepared nanoparticles will increase as well. In this regard, novel MVL5 is selected as the ionizable cationic lipid in the present invention. One MVL5 molecule contains a multivalent cationic amino group, compared with ionizable cationic lipids containing monovalent cationic amino groups (such as EDOPC or DOTAP), in the process of preparing the lipid nanoparticles, less cationic lipids can achieve high mRNA cell transfection efficiency and significantly reduced cytotoxicity.

[0063] Successfully escaping from late endosomes is the key to drug delivery by the intracellular delivery system, which can prevent the drug molecules from being degraded by a large number of enzymes in the late endosomes. Related studies have shown that phosphatidylethanolamine combines with different kinds of unsaturated aliphatic hydrocarbons to form a phospholipid auxiliary lipid (such as DOPE). The phospholipid auxiliary lipid is negative in a neutral physiological environment (pH 7.4) with layered spatial structures. When the pH decreases (pH 5.0-6.0), phosphoethanolamine (PE) protonation makes the spatial conformation of the complex into hexagonal. The hexagonal complex is more destructive to the late endosome membrane. Taking advantage of this property, the phospholipid auxiliary lipid can help lipid nanoparticles escape from late endosomes under acidic conditions and prevent mRNA from being degraded by enzymes in the late endosomes.

[0064] DSPE-PEG2000 can increase the hydrophilicity of lipid nanoparticles, reduce the non-specific adsorption of lipid nanoparticles to proteins, and lower the probability of phagocytosis by mononuclear macrophages, because of its unique amphiphilic properties and spatial configuration.

[0065] In sum, in the present invention, components and their proportion in lipid nanoparticles are selected according to the sensitivity to pH changes, membrane stability, mRNA transfection efficiency, and loading effect of mRNA molecules, and the specific implementation paths for preparation, purification and concentration of the mRNA/lipid nanoparticles are optimized, aiming at developing an efficient targeted intracellular delivery system for mRNA nucleic acid drugs that can be used in large-scale clinical applications.

[0066] I. Main Reagents

TABLE-US-00001 Main reagents in the present invention Supplier DOTMA Avanti Polar 1,2-di-O-octadecenyl-3-trimethylammonium propane Lipids (chloride salt) EDOPC Avanti Polar 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine Lipids MVL5 Avanti Polar N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino- Lipids propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide DOTAP Avanti Polar 1,2-dioleoyl-3-trimethylammonium-propane(chloride Lipids salt) DMAP-BLP Avanti Polar 3-(dimethylamino)propyl(12Z,15Z)-3-[(9Z,12Z)- Lipids octadeca-9,12-dien-1-yl]henicosa-12,15-dienoate DC-Cholesterol•HCl Avanti Polar (3β-[N-(N′,N′-dimethylaminoethane)- Lipids carbamoyl]cholesterol hydrochloride) Dlin-KC2-DMA SuperLan 2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4- yl)-N,N-dimethylethanamine DOPS Avanti Polar (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) Lipids DOPE Avanti Polar (1,2-di-(9Z-octadecenoyl)-sn-glycero-3- Lipids phosphoethanolamine) DMPC Avanti Polar (1,2-Dimyristoyl-sn-glycero-3-PC) Lipids DOPC Avanti Polar 1,2-dioleoyl-sn-glycero-3-phosphocholine Lipids DSPE-PEG 2000 Avanti Polar 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- Lipids [methoxy(polyethylene glycol)-2000] PEG-DMG 2000 Avanti Polar 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene Lipids glycol-2000 C14-PEG2000 Avanti Polar 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- Lipids [methoxy (polyethylene glycol)-2000] (ammonium salt) Cholesterol Avanti Polar Lipids Sodium citrate Sigma-Aldrich Sodium acetate Sigma-Aldrich RNase free deionized water Thermo Fisher Scientific Chloroform Sigma-Aldrich anhydrous ethanol Sigma-Aldrich 10xPBS (pH 7.4) Sigma-Aldrich 1xTris-borate-EDTA (TBE) buffer BioRad Gelred nucleic acid gel dye Biotium 6xloading Dye Biotium Agarose BIOWEST NaH.sub.2PO.sub.4•H.sub.2O Sigma-Aldrich Na.sub.2HPO.sub.4•7H.sub.2O Sigma-Aldrich Quant-iT ™ RiboGreen ™ RNA Assay Kit Thermo Fisher Scientific Triton-X100 Sigma-Aldrich

[0067] II. Main Instruments and Consumables

TABLE-US-00002 Main instruments and consumables in the present invention Supplier NanoAssemblr ® Benchtop nanoparticle synthesis Precision system Nanosystem Rotary evaporator (R-1020) Great Wall Temperature control device (DL10-3000) Great Wall Vacuum acquisition and control device (SHB-B95) Great Wall Microplate reader (Infinite M200 Pro NanoQuant) TECAN Dynamic light scattering nanoparticle size analyzer Malvern (Zetasizer Pro) Panalytical Gel Imager (GelDoc XR+) Bio-Rad Flow cytometry (FACSCanto) BD Biosciences Inverted fluorescence microscope (CKX53) Olympus Vortex oscillator SITIME Magnetic stirrer SITIME Pipette Eppendorf −20° C. low temperature refrigerator Zhongke Meiling 4° C. low temperature refrigerator Zhongke Meiling −80° C. ultra-low temperature refrigerator Thermo Fisher Scientific Microwave oven Zhongke Meiling Nucleic acid electrophoresis system BioRad Filter tip Axygen Centrifuge tube Axygen Round bottom flask (25 ml) Great Wall Dialysis membrane Thermo Fisher Scientific Ultrafiltration centrifuge tube EMD Millipore Erlenmeyer flask (200 ml) Great Wall

[0068] III. Experimental Methods

[0069] 1. Formation and Drying of Thin Lipid Membranes

[0070] Firstly, the four components (MVL5, DOPE, cholesterol, and DSPE-PEG 2000) were completely dissolved in chloroform, respectively, and then the dissolved solutions respectively containing 25 mg, 41.5 mg, 50.68 mg, and 14.06 mg of the four components (with a molar ratio of 10:26:61.5:2.5) were mixed uniformly, and then moved into a 25 mL round bottom flask for a slow rotary evaporation at 0.06 Mpa (gauge pressure) and 32° C. to remove chloroform until a layer of thin lipid membrane with uniform thickness is formed at the bottom of the round bottom flask. Subsequently, a vacuum drying is carried out at −0.1 Mpa (gauge pressure) and 28° C. for 5 h (to completely remove the residual chloroform).

[0071] 2. Thin Lipid Membrane Dissolved in Anhydrous Ethanol

[0072] 10 mL of anhydrous ethanol was added to the round bottom flask, and then the round bottom flask was moved to the magnetic stirrer and stirred evenly for 30 min until the thin lipid membrane disappeared (the thin lipid membrane was completely dissolved in the anhydrous ethanol).

[0073] 3. Dilution of mRNA Solution

[0074] mRNA (100 μg/mL) dissolved in RNase-free deionized water was diluted with a sodium citrate buffer with a concentration of 50 mM and a pH of 4.0.

[0075] 4. Preparation of mRNA/Lipid Nanoparticle Suspension Solution

[0076] The mixed components completely dissolved in anhydrous ethanol were quickly mixed with the diluted mRNA solution, which was controlled by Microfluidic mixers (NanoAssemblr) (operated by the software PRECISION Nanosystems thereof). The flow rate ratio (FRR) of ethanol phase to water phase was 1:3, and their flow rates were 5 mL/min and 15 mL/min, respectively. The total flow rate (TFR) in the mixing pipeline was 12 mL/min. The suspension solution of ethanol and water was obtained (the mass ratio of mRNA to ionizable cationic liposomes was 1:12.5 (w/w)).

[0077] 5. Purification and Concentration of mRNA/Lipid Nanoparticles

[0078] The ethanol was removed by dialysis. The suspension solution obtained by the above step was dialyzed twice in PBS with a volume 200 times the volume of suspension solution and a pH of 7.4 (with 10 kd dialysis membrane). The first dialysis was performed at room temperature (25° C.) for 3 h, and the second dialysis was performed at 4° C. for 15 h. The mRNA/lipid nanoparticle suspension solution without ethanol was concentrated by centrifugal ultrafiltration to reach the final concentration of 1 μg/μl. The mRNA/lipid nanoparticle suspension solution was filtered by 0.22 μm membrane for 5 times and then filtered by 0.1 μm membrane for 3 times, and subsequently, was sub-packaged and preserved at −80° C.

[0079] IV. Experimental Results

[0080] 1. Particle Size of Prepared mRNA/Lipid Nanoparticles

[0081] Different batches of samples filtered by 0.22 μm and 0.1 μm filter membranes were measured by the dynamic light scattering nanoparticle size analyzer. It was found that the average particle size of the mRNA/lipid nanoparticles was 85 nm. In a neutral environment (pH 7.4), the Zeta potential of the mRNA/lipid nanoparticles was +32.6 mV.

[0082] 2. pH Sensitivity and Specificity Analysis of mRNA/Lipid Nanoparticles

[0083] The mRNA/lipid nanoparticles were mixed with phosphate buffer (PB) solutions of different pH and incubated at 37° C. for 30 min. The potential change of the mixture was measured by Zeta potentiometer. By measuring the surface potential changes of lipid nanoparticles at different pH, the stability of the mRNA/lipid nanoparticles in a neutral environment was reflected, and the ability of mRNA in the mRNA/lipid nanoparticles on escaping from late endosomes in the acidic environment was shown. The experimental results showed that the Zeta potential of the prepared mRNA/lipid nanoparticles was relatively stable in a neutral environment, but the Zeta potential of the surface of the mRNA/lipid nanoparticles increased sharply in an acidic environment. The results were shown in FIG. 2.

[0084] 3. Qualitative Analysis of Encapsulation Efficiency of mRNA/Lipid Nanoparticles

[0085] (1) An appropriate amount of agarose was weighed and added into an appropriate amount of 1×TBE buffer to prepare 0.7% agarose nucleic acid gel.

[0086] (2) Appropriate amounts of the mRNA/lipid nanoparticle suspension solution and unencapsulated free mRNA were added into 6×loading Dye loading buffer, and then mixed and added into sample wells (with 250 ng of mRNA in each well). After adding the samples, the electrophoresis tank was covered and the power supply was turned on. The voltage of the power supply was controlled to maintain 60 V and the current was maintained above 40 mA. When the bromophenol blue band moved to about 2 cm from the front of the gel, the power supply was turned off and the electrophoresis was stopped.

[0087] (3) After the electrophoresis, the nucleic acid gel was moved into a Gelred nucleic acid dye solution having a concentration of 0.5 μg/ml and stained in a dark environment at room temperature for 25 min. After staining, the gel was moved into the gel imager, and the stained mRNA was observed and photographed under ultraviolet light with the wavelength of 254 nm. The results showed that in the control group of unencapsulated free mRNA, there was mRNA staining in the electrophoresis lane (The free mRNA is shown in the box), while the mRNA encapsulated in the lipid nanoparticles (mRNA/LNPs) was completely blocked in the sample wells. (The results are shown in FIG. 3).

[0088] 4. Accurate Quantitative Analysis of Encapsulation Efficiency of mRNA/Lipid Nanoparticles

[0089] (1) The prepared mRNA/lipid nanoparticle suspension solution and PBS (negative control, having the same volume of TE buffer) was diluted to 4 ng/μL with TE buffer in the kit to obtain an mRNA/lipid nanoparticle working solution.

[0090] (2) The mRNA/lipid nanoparticle working solution was further diluted with TE buffer (or TE buffer containing 2% of Triton-X100) to reduce its concentration to half, and mixed, and then kept at 37° C. for 10 min (TE buffer without Triton-X100 was used for the determination of unencapsulated free mRNA, while TE buffer containing 2% of Triton-X100 was used for the determination of the total mRNA in the mRNA/lipid nanoparticle working solution, where the total mRNA included the free mRNA and the mRNA encapsulated in the lipid nanoparticles). Each group of samples was set for three repetitions.

[0091] (3) After obtaining the standard curve of fluorescence intensity/concentration by calibrating with the standard sample, an appropriate amount of Quanti-iT™ RiboGreen RNA reagent nucleic acid dye was added to each group of samples for staining for 5 min according to the instructions of the kit. Each group of samples after dyeing was moved to the TECAN microplate reader for detection, and the software I-Control v.3.8.2.0 was used to accurately quantify the mRNA in the samples.

[0092] (4) The following formula was used to calculate the encapsulation efficiency of the mRNA in the lipid nanoparticles

Encapsulation efficiency=[1−m(free mRNA)/m(total mRNA)]×100%].

[0093] By measuring the concentrations of the free mRNA and total mRNA in three repeatedly diluted samples, the results showed that the encapsulation efficiency of the mRNA in the mRNA/lipid nanoparticles (mRNA/LNPs) prepared by this method was more than 98%.

[0094] 5. mRNA Intracellular Transfection Efficiency of mRNA/Lipid Nanoparticle Drug Delivery System

[0095] DC2.4 cells were inoculated into a 24-well plate (3×10.sup.5 cells/well). Free eGFP-mRNA (0.5 μg) and the mRNA/lipid nanoparticles (0.5 μg) were added to the cell culture medium, three wells for each group. After culturing for 48 h, the expression of the eGFP-mRNA in the DC2.4 cells was detected by the fluorescence microscope (the results are shown in FIG. 4A) and flow cytometry (the results are shown in FIG. 4B). These results showed that compared with unencapsulated free mRNA, the encapsulated mRNA can be effectively mediated into the cells and expressed at a high level by the mRNA/lipid nanoparticle (mRNA/LNPs) drug delivery system.

[0096] The preferred implementation ways and embodiments of the present invention are described in detail above, but the present invention is not limited to the above implementation ways and embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can also be made without departing from the conception of the present invention.