CN115844833A - Ionizable lipid nanoparticle and preparation method thereof - Google Patents

Abstract

The invention discloses an ionizable lipid nanoparticle, which is prepared from neutral lipids, ionizable lipids, phospholipids, and PEG-lipids. The preparation method includes dissolving raw materials in ethanol and mixing them to obtain ethanol-lipids The lipid mixture was mixed with the lipid mixture and aqueous buffer by microfluidics to obtain a final lipid concentration of 0.6‑1.8mg/mL. The invention belongs to the technical field of preparation of lipid nanoparticles, and specifically provides a greatly improved particle size dispersion, and after preparation of lipid vesicles, it can be mixed with different types of nucleic acids for immediate use, improving the efficiency of nucleic acid High delivery efficiency, can be used in a wide range of application scenarios such as in vitro cell or in vivo transfection, while ensuring the reliability of the experiment and the continuity of the later stage, with better particle size controllability and higher reliability of ionizable lipids for nucleic acid delivery Nanoparticles and methods for their preparation.

Description

A kind of ionizable lipid nanoparticle and preparation method thereof

technical field

The invention belongs to the technical field of preparation of lipid nanoparticles, and relates to a method of using the first step of a two-step method to prepare universal lipid particles in vitro and in vivo to deliver nucleic acid molecules and achieve transfection effects, specifically an ionizable Lipid nanoparticles and methods for their preparation.

Background technique

Therapeutic and diagnostic systems based on nanoparticles (NPs) have been extensively studied, many of which have been approved for clinical use. Currently, an increasing number of NPs with diverse chemical, physical, and biological properties are being fabricated with the aim of controlling their fate in organisms and addressing multiple in vivo delivery barriers.

The preparation process of ionizable lipid vesicles is considered as a simple water-oil phase mixing in the traditional technology, but it is difficult to ensure that the particle size dispersion is within a reasonable range. At present, most nucleic acid drugs in clinical practice use liposome technology, which proves the safety of this technology. caused by drug instability. In view of the above problems, the technology provided by this solution can help solve such problems.

Compared with other nucleic acid in vitro transfection preparations, ionizable lipid vesicles are more suitable for in vitro cell transfection under high uniformity. The cytotoxicity of the staining reagent is high, while the liposome is low toxicity and high efficiency. It is concluded that the invented two-step lipid vesicle method can improve the rapid push of nucleic acid molecules into animal experiments.

In view of this, the present invention is proposed.

Contents of the invention

In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a preparation technology that can test the effectiveness of nucleic acid molecules in vitro and is suitable for in vivo transfection, simplifies the process of drug development from drug screening to animal experiments, and provides a A lipid vesicle that delivers nucleic acid with higher reliability, improves the delivery efficiency of nucleic acid, and can be used in a wide range of application scenarios such as in vitro cell or in vivo transfection and a preparation method thereof.

The technical scheme adopted by the present invention is as follows: an ionizable lipid nanoparticle of the present invention is prepared from neutral lipids, ionizable lipids, phospholipids, and PEG-lipids.

As a preferred version, the neutral lipid is cholesterol;

The ionizable lipid is Dlin-MC3-DMA;

The phospholipid is one or more of DOPC and DSPC;

The PEG-lipid is one or more of PEG2000-DMG, PEG5000-DMG, PEG2000-DMA, PEG5000-DMA.

Further, the PEG lipid includes PEG2000 or PEG5000; preferably, the first PEG lipid is PEG2000.

Further, the molar ratio range of ionizable lipid: neutral lipid: PEG-lipid is 50-90%: 10-50%: 0.1-5%, or ionizable lipid: phospholipid: neutral Lipid:PEG-lipid molar ratios ranged from 30-60%:4-20%:35-55%:0.1-5%.

In a preferred scheme, the molar ratio is 50% of ionizable lipids, 10% of phospholipids, 38% of neutral lipids, and 2% of PEG-lipids.

As a further elaborated scheme, this scheme also discloses a preparation method of ionizable lipid nanoparticles, comprising the following steps:

S1: Prepare the raw materials with corresponding molar mass ratio, the raw materials include ionizable lipids, phospholipids, neutral lipids and PEG-lipids, and then the ionizable lipids, phospholipids, neutral lipids and PEG-lipids Substances were dissolved in alcohol solution;

S2: mixing several raw materials respectively dissolved in the alcohol solution to obtain the lipid mixture in the alcohol solution;

S3: Add the lipid mixture to the aqueous buffer solution, the volume of the aqueous buffer solution is 3 to 5 times that of the lipid mixture, to obtain 10-33.3% (vol lipid solution/vol aqueous solution) and 0.6-1.8 mg/mL ( Amount of lipid divided by total volume after microfluidic mixing) Concentration of final lipid.

Further, in the above steps, a microfluidic pre-formed vesicle method is used to generate nanoparticles containing the ionizable lipid.

In a preferred solution, the alcohol solution includes one of methanol, ethanol, and acetone, and the volume ratio of the alcohol solution is 70%-99.9%, preferably ethanol with a volume ratio of 97%.

In a preferred version, the aqueous buffer is 10-100mM phosphate buffer, 10-100mM tris buffer, or 10-100mM sodium acetate buffer, with a pH of 6.3-6.5, preferably phosphate buffer .

Further, in the step S3, the ethanol can be volatilized by placing at room temperature.

In a further scheme, the obtained lipid vesicles are injected into a ready-to-use dialysis tube (less than 50k) and dialyzed at 4°C for 24 hours, and the buffer is replaced 3-5 times;

In a preferred solution, the stabilized lipid vesicles can be selectively size-fractionated by hollow fiber tangential flow filtration or a centrifuge, and purified to reduce particle size dispersion, so that the particle size is 80-120nm or 180nm-220nm, and the particle size is dispersed The density is less than 0.1, and the PEG concentration on the surface is 20PEG/100nm ² ;

In the preferred solution, in the step S3, a microfluidic human-type micron Y-shaped channel is used, and buffer and ethanol-lipid mixture are injected at both ends; the microfluidic chip is 60-120 μm high, 100-200 μm wide, and the middle Distribution 20-31 μm high, 30-50 μm thick chevron baffles, where the volume of aqueous buffer is 3 to 5 times that of lipid mixture (10-33.3% vol lipid solution/vol aqueous solution), buffer is preferred 10mM phosphate buffer, pH 6.5. The volume ratio of ethanol-lipid to buffer is preferably 33%. The microfluidic injection rate is 3-10 mL/min, preferably 3 mL/min.

Further, the pH of the final lipid vesicle product in the aqueous buffer should be similar to the pKa of the ionizable lipid, and the neutral ionizable lipid combines with phospholipids and neutral lipids in a lipophilic manner to form a stable Core structure, PEG-lipid PEG forms a hydrophilic layer on the surface to protect the lipid structure and achieve thermodynamic equilibrium.

In this scheme, nucleic acid molecules will be encapsulated into such vesicle particles, and the mixed solution of this scheme contains ethanol with a concentration of 10% (v/v)-40% (v/v); meanwhile, in different lipids In the case of the combination of cytoplasmic vesicles, the pH of the buffer will be adjusted to 3-5, which is suitable for encapsulating mRNA; in the process of mixing buffer and ethanol, it is necessary to warm the ethanol-buffer mixture to room temperature.

Preferably, the lipid vesicles are added to the ethanol-buffer mixture, mixed evenly, and then placed at room temperature for 10-30 minutes, then the target nucleic acid solution is added, mixed evenly, and then allowed to stand for 10-30 minutes;

The buffer is 10mM phosphate buffer;

The pH of the buffer is 3-5, preferably 4;

The ethanol volume ratio is 10%-40%, preferably the ethanol volume ratio is 30%;

The mass ratio of the nucleic acid molecule to the lipid is 1: (5-30), preferably the mass ratio of the nucleic acid molecule to the lipid is 1:20;

The mRNA is mcherry and eGFP.

Preferably, the lipid vesicles are loaded with nucleic acid molecules to become mature and stable final liposomes, the mature liposomes are injected into ready-to-use dialysis tubes, dialyzed for 3 hours at low temperature, and the phosphate buffer solution with a pH of 4 is replaced 3 times, 99.9% of ethanol can be removed; the final product can be selectively passed through a 0.45μm filter membrane to reduce the particle size dispersion, and the final particle size dispersion is less than 0.2.

Finally, add the encapsulated liposome-nucleic acid complex into the dialysis tube, remove the ethanol, or let the ethanol evaporate at room temperature, and then store it at a low temperature of 4°C.

A kind of ionizable lipid nanoparticle and preparation method thereof of this scheme, adopt the beneficial effect that the present invention obtains of above-mentioned scheme as follows:

1. The traditional one-step liposome preparation is divided into two steps. First, lipid "vesicles" are prepared, and then drug molecules are loaded. The synthesis of lipid vesicles is a simplified lipid composition, so that the synthesized nanoparticles have Better particle size controllability, and physical and chemical stability.

2. Combining a mixture of lipids with a buffered aqueous solution of nucleic acids to produce an intermediate mixture containing nucleic acids encapsulated in lipid particles, wherein said encapsulated nucleic acids are present at a nucleic acid/lipid ratio of about, preferably 3 to 10 wt % , the composition ratio of lipids in lipid vesicles is very different from that in conventional liposomes. The intermediate mixture may optionally be size fractionated to obtain lipid-encapsulated nucleic acid particles.

3. When synthesizing liposome vesicles, the pH of the buffer is similar to the pKa of the ionizable lipids, and the neutral ionizable lipids combine with phospholipids and neutral lipids in a lipophilic manner to form a stable core structure. PEG-lipid PEG forms a hydrophilic layer on the surface to protect the lipid structure, achieves thermodynamic equilibrium, and can quickly achieve high-uniform particle size dispersion; lipid vesicles with high particle size uniformity, even after carrying nucleic acid molecules , the particle size dispersion remains below 0.2. Nucleic acid transfectability based on this is more reliable.

4. The preparation of lipid vesicles is prepared by microfluidic method. Compared with the traditional film method and ultrasonic method, the particle size dispersion can be controlled below 0.1, and the particle size can still be kept below 0.2 after the mRNA is loaded in the later stage. Comply with important international parameters such as FDA.

5. The lipid vesicles produced by this method have a specific particle size range and surface PEG concentration. Compared with traditional liposomes, the lipid vesicles obtained by this method have higher in vivo and in vitro transfection after carrying mRNA. It can actively target the liver or actively not target the liver, which can improve the therapeutic window for specific disease areas.

6. The method of carrying nucleic acid is to lower the pH, so that the ionizable lipid is positively charged, thereby attracting negatively charged nucleic acid molecules for encapsulation. The No. 2 solution ethanol in this product can accelerate the fluidity of the lipid molecules in the vesicles that have been formed, and better include the attracted nucleic acid molecules into the nanobody. The purpose of removing ethanol is to allow lipid or lipid-nucleic acid complexes to coagulate more and improve product stability.

7. In addition to the low toxicity of liposomes, the product can be mixed with specific mRNA before administration according to the actual application needs, without pre-compounding with mRNA to make nanoparticles and other operations, making its application more flexible and most importantly The most important thing is the precise control of particle size, which strengthens the reliability of experimental data.

8. Secondly, it solves the high toxicity of traditional infectious reagents and cannot be directly applied to animal experiments, and at the same time improves the reliability of transfection from in vitro cells to in vivo.

Description of drawings

Fig. 1 is the lipid nanoparticle diameter under the standard synthesis condition in this scheme;

Fig. 2 is the example of liposome vesicle in embodiment 1 after carrying the nucleic acid molecule of eGFP, compare lipo in vitro cell transfection efficiency at the same concentration;

Fig. 3 is the example of liposome vesicle in embodiment 2 after carrying the nucleic acid molecule of mcherry, comparing lipo at the same concentration in vitro cell transfection efficiency;

Figure 4 is an illustration of the mRNA and short-chain gRNA that encapsulate Cas9 in lipid vesicles in Example 3, and compare the protein that encapsulates Cas9 with gRNA;

Figure 5 is the mcherry nucleic acid sequence;

Figure 6 is the nucleic acid sequence of eGFP.

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, and are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them; based on The embodiments of the present invention and all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

As shown in Figures 1-6, an ionizable lipid nanoparticle of the present invention is prepared from neutral lipids, ionizable lipids, phospholipids, and PEG-lipids.

As a preferred version, the neutral lipid is cholesterol;

The ionizable lipid is Dlin-MC3-DMA;

The phospholipid is one or more of DOPC and DSPC, preferably DSPC;

The PEG-lipid is one or more of PEG2000-DMG, PEG5000-DMG, PEG2000-DMA, PEG5000-DMA, preferably PEG2000-DMG.

This program also discloses a preparation method of ionizable lipid nanoparticles, comprising the following steps:

S1: Prepare raw materials with corresponding molar mass ratios, the raw materials include 50% ionizable lipids, 10% phospholipids, 38% neutral lipids, and 2% PEG-lipids in molar ratios, and then mix the above ionizable lipids, phospholipids Substance, neutral lipid and PEG-lipid were dissolved in 97% ethanol respectively;

S2: mixing several raw materials respectively dissolved in 97% ethanol to obtain a lipid mixture in ethanol;

S3: Add lipid mixture to 10mM phosphate buffer at pH 6.5, mix lipid mixture and aqueous buffer by microfluidic method, microfluidic human micron Y-shaped channel, inject phosphate buffer at both ends and ethanol-lipid mixture, the microfluidic injection rate is 3mL/min, in which the microfluidic chip is 60-120μm high, 100-200μm wide, the middle distribution is 20-31μm high, and the chevron baffle is 30-50μm thick , wherein the volume of the aqueous buffer is 3 to 5 times that of the lipid mixture (10-33.3% vol lipid solution/vol aqueous solution), the volume of the aqueous buffer is 3 to 5 times the lipid mixture, respectively to obtain 10- Final lipid at a concentration of 33.3% (vol lipid solution/vol aqueous solution) and 0.6-1.8 mg/mL (lipid mass divided by total volume after microfluidic mixing).

The obtained lipid vesicles are injected into a ready-to-use dialysis tube, preferably with a filter pore size of 25k, dialyzed at 4°C for 24 hours, and the buffer is changed 3-5 times, preferably the buffer is 10 mM phosphate buffer, pH 6.5. The final lipid vesicle No. 1 reagent product was obtained after dialysis.

The product also includes No. 2 reagent, preferably used to mix No. 1 product with the target nucleic acid molecule. Reagent No. 2 is preferably 20 mM phosphate buffer, pH 3, and contains preferably 20% ethanol by volume.

The function of this product is to efficiently carry mRNA molecules, preferably mcherry and eGFP (sequences shown in Figure 5 and Figure 6 below), into the liposome vesicles in the No. 1 reagent. The specific operation is to mix the No. 1 product reagent with the No. 2 buffer solution reagent at room temperature, and let it stand for 10-30 minutes. Afterwards, the target nucleic acid molecules are added to the reagent, mixed evenly, and left to stand for 10-30 minutes, then more than 95% of the nucleic acid molecules can be successfully loaded. The preferred mass ratio of nucleic acid to liposome vesicle is 1:4, wherein the volume ratio of nucleic acid is less than 5%.

Finally, add the encapsulated lipid vesicle-nucleic acid complex (that is, mature liposome) into a dialysis tube to remove ethanol, or place it at room temperature to allow the ethanol to evaporate, and then store it at a low temperature of 4 degrees.

As shown in Figure 1, it is the particle size of lipid vesicles under standard synthesis conditions. The example in the figure shows the particle size and particle size dispersion of 9 kinds of lipids with different composition and ratio at different synthesis rates. The 9 ingredients are

Ionizable lipid:neutral lipid:PEG-lipid ratio, 50%:45%:5%, 60%:35%:5%, 70%:25%:5%,

Alternatively the ionizable lipid:phospholipid:neutral lipid:PEG-lipid ratio ranges from 50%:10%:38.5%:1.5%.

50%: 10%: 39%: 1%, 50%: 10%: 39.5%: 0.5%,

The microfluidic injection rates were 3, 5, and 8 mL/min, respectively.

Example 1, as shown in Figure 2, the ratio range of ionizable lipid: phospholipid: neutral lipid: PEG-lipid is 50%: 10%: 38.5%: 1.5%, microfluidic injection Under the condition of a rate of 3mL/min, after liposome vesicles are loaded with eGFP nucleic acid molecules, an example of comparing the in vitro cell transfection efficiency of lipofectamine (lipo) at the same concentration. Liposome vesicles showed higher transfection efficiency, lipo showed certain cytotoxicity at this concentration, and HEK395 was used for cells.

Example 2, as shown in Figure 3, the ratio range of ionizable lipid: phospholipid: neutral lipid: PEG-lipid is 50%: 10%: 38.5%: 1.5%, microfluidic injection Under the condition of a rate of 3 ml per minute, the liposome vesicles are loaded with mcherry nucleic acid molecules, and an example of comparing lipo transfection efficiency in vitro at the same concentration. Liposome vesicles showed higher transfection efficiency, and lipo showed certain cytotoxicity. HEK395 cells were used.

Example 3, as shown in Figure 4, uses lipid vesicles to encapsulate Cas9 mRNA and short-chain gRNA, and compares the protein that encapsulates Cas9 plus gRNA. Lipid vesicles exhibited equally excellent cell editing. HEK395 cells were used.

In this scheme, when synthesizing lipid vesicles, the pH of the buffer is similar to the pKa of the ionizable lipids, and the neutral ionizable lipids combine with phospholipids and neutral lipids in a lipophilic manner to form a stable core structure. PEG -Lipid PEG forms a hydrophilic layer on the surface to protect the lipid structure, achieve thermodynamic equilibrium, and quickly achieve high uniform particle size dispersion.

In the methods described herein, a mixture of lipids is combined with a buffered aqueous solution of nucleic acids to produce an intermediate mixture containing nucleic acids encapsulated in lipid particles, wherein the encapsulated nucleic acids comprise about, preferably 3 to 10 wt% nucleic acids. /lipid ratio exists. The intermediate mixture may optionally be size fractionated to obtain lipid-encapsulated nucleic acid particles, wherein the lipid fraction is a vesicle preferably having a diameter of 80-120 nm, or preferably about 180-220 nm.

The method of loading nucleic acids is to lower the pH so that the ionizable lipids are positively charged, thereby attracting negatively charged nucleic acid molecules for encapsulation. The No. 2 solution ethanol in this product can accelerate the fluidity of the lipid molecules in the vesicles that have been formed, and better include the attracted nucleic acid molecules into the nanobody. The purpose of removing ethanol is to allow lipid or lipid-nucleic acid complexes to coagulate more and improve product stability.

In addition to the low toxicity of liposomes, the product can be mixed with specific mRNA before administration according to the actual application needs, without pre-compounding with mRNA to make nanoparticles and other operations, making its application more flexible, and most importantly Precise control of particle size enhances the reliability of experimental data.

Secondly, under the preference of lipid vesicles with a particle size of 180 to 220 nm, the transfection efficiency of in vitro cells is much higher than that of ordinary particle size, that is, 100 nm. Bring indicative direction for the application of liposome in the future.

The research and development design of the preparation process is aimed at: first, the stability of the transfection reagent at this stage is poor, and the optimization time required is long; second, the liposome product at this stage has poor stability and high particle size dispersion. Compared with the existing transfection reagents on the market, this product is more stable, and it is easier to be used as a standard reference for in vitro cell effectiveness, and can be directly used in preclinical animal experiments; compared with the same type of nano products on the market, its particle size Smaller diameter dispersion, less cytotoxic side effects, and lower storage environment requirements; when used, it can be directly matched with specific mRNA sequences and used flexibly to meet different application requirements.

It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is a relationship between these entities or operations. There is no such actual relationship or order between them. Furthermore, the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus comprising a set of elements includes not only those elements, but also includes elements not expressly listed. other elements of or also include elements inherent in such a process, method, article, or device.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

The present invention and its implementations have been described above, and this description is not limiting. What is shown in the drawings is only one of the implementations of the present invention, and the actual structure is not limited thereto. All in all, if a person of ordinary skill in the art is inspired by it, and without departing from the inventive concept of the present invention, without creatively designing a structure and an embodiment similar to the technical solution, it shall fall within the scope of protection of the present invention.

Claims (10)

1. An ionizable lipid nanoparticle, characterized in that: it is prepared from neutral lipids, ionizable lipids, phospholipids, and PEG-lipids. 2. A kind of ionizable lipid nanoparticle according to claim 1, characterized in that: the neutral lipid is cholesterol; The ionizable lipid is Dlin-MC3-DMA; The phospholipid is one or more of DOPC and DSPC; The PEG-lipid is one or more of PEG2000-DMG, PEG5000-DMG, PEG2000-DMA, PEG5000-DMA. 3. a kind of ionizable lipid nanoparticle according to claim 2, is characterized in that: described ionizable lipid: neutral lipid: the molar ratio scope of PEG-lipid is in 50-90%: 10- 50%:0.1-5%; or ionizable lipid:phospholipid:neutral lipid:PEG-lipid molar ratio ranges from 30-60%:4-20%:35-55%:0.1-5% . 4. A kind of ionizable lipid nanoparticle according to claim 3, characterized in that: the molar ratio of the ionizable lipid: phospholipid: neutral lipid: PEG-lipid is 50%: 10% :38% :2%. 5. A method for preparing an ionizable lipid nanoparticle according to any one of claims 1-4, comprising the following steps: S1: Prepare the raw materials with corresponding molar mass ratio, the raw materials include ionizable lipids, phospholipids, neutral lipids and PEG-lipids, and then the ionizable lipids, phospholipids, neutral lipids and PEG-lipids Substances are respectively dissolved in the alcohol solution; the alcohol solution includes one of methanol, ethanol and acetone, and the volume ratio of the alcohol solution is 70%-99.9%; S2: Mix the several raw materials respectively dissolved in the alcohol solution to obtain the lipid mixture in the alcohol solution; the aqueous buffer is 10-100mM phosphate buffer, 10-100mM tris buffer, or 10-100mM sodium acetate buffer, pH 6.3-6.5; S3: Add lipid mixture to aqueous buffer, mix lipid mixture and aqueous buffer by microfluidic method, microfluidic chip is 60-120 μm high, 100-200 μm wide, middle distribution 20-31 μm high, 30- 50 μm thickness of the herringbone baffle, the microfluidic injection rate of 3-10mL/min, the volume of the aqueous buffer is 3 to 5 times that of the lipid mixture, respectively to obtain 10-33.3% (vol lipid solution / vol aqueous solution ) and a final lipid concentration of 0.6-1.8mg/mL can be left at room temperature to allow ethanol to evaporate. 6. A method for preparing ionizable lipid nanoparticles according to claim 5, characterized in that: the obtained lipid vesicles are injected into a ready-to-use dialysis tube and dialyzed at 4°C for 24 hours, and the buffer solution is replaced for 3-5 hours. Second; the stabilized lipid vesicles can be selectively size-graded by hollow fiber tangential flow filtration or centrifuge, purified to reduce particle size dispersion, so that the particle size is 80-120nm or 180nm-220nm, and the particle size dispersion is less than 0.1, the PEG concentration on the surface is 20PEG/100nm ² . 7. the preparation method of a kind of ionizable lipid nanoparticle according to claim 6 is characterized in that: the pH of final lipid vesicle product in aqueous buffer solution should be approximate with the pKa of ionizable lipid, Non-toxic ionizable lipids combine with phospholipids and neutral lipids in a lipophilic manner to form a stable core structure, and PEG-lipid PEG forms a hydrophilic layer on the surface to protect the lipid structure and achieve thermodynamic equilibrium. 8. the preparation method of a kind of ionizable lipid nanoparticle according to claim 7, is characterized in that: nucleic acid molecule will be encapsulated in this type of vesicle particle, and the mixed solution of this scheme contains concentration and is 10% ( v/v)-40% (v/v) ethanol; at the same time, under the combination of different lipid vesicles, the pH of the buffer will be adjusted to 3-5, which is suitable for encapsulating mRNA; in the mixed buffer and During the ethanol process, the ethanol-buffer mixture needs to be warmed to room temperature. 9. The preparation method of a kind of ionizable lipid nanoparticles according to claim 8, characterized in that: the lipid vesicles are added to the ethanol-buffer mixed solution and mixed uniformly, then placed at room temperature for 10-30min, Then add the target nucleic acid solution, mix well and let stand for 10-30min; The buffer is 10mM phosphate buffer; The pH value of the buffer solution is 3-5; The ethanol volume ratio is 10%-40%; The mass ratio of the nucleic acid molecule to the lipid is 1: (5-30); The mRNA is mcherry and eGFP. 10. the preparation method of a kind of ionizable lipid nanoparticle according to claim 9, is characterized in that: described lipid vesicle becomes mature stable final liposome after carrying nucleic acid molecule, injects mature liposome Ready-to-use dialysis tubing, dialyzate at low temperature for 3 hours, replace the phosphate buffer with pH 4 for 3 times, and remove 99.9% of ethanol; the final product can optionally pass through a 0.45μm filter membrane to reduce particle size dispersion, The final particle size dispersion is less than 0.2.

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