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

Abstract

The invention discloses an ionizable lipid nanoparticle which is prepared from neutral lipid, ionizable lipid, phospholipid and PEG-lipid, and the preparation method comprises the steps of dissolving raw materials in ethanol and mixing to obtain an ethanol-lipid mixture, and mixing the lipid mixture and an aqueous buffer solution by a microfluidic method to obtain final lipid with the concentration of 0.6-1.8 mg/mL. The invention belongs to the technical field of preparation of lipid nanoparticles, and particularly provides ionizable lipid nanoparticles for delivering nucleic acid, which greatly improve the particle size dispersity, can be prepared with different types of nucleic acid after lipid vesicles are prepared, improve the delivery efficiency of the nucleic acid, can be used in wide application scenes such as in-vitro cell or in-vivo transfection and the like, ensure the reliability and the later continuity of an experiment, and have better particle size controllability and higher reliability, and a preparation method thereof.

Description

Ionizable lipid nanoparticle and preparation method thereof

Technical Field

The invention belongs to the technical field of preparation of lipid nanoparticles, relates to a method for preparing in-vitro and in-vivo universal lipid particles by using the first step of a two-step method to deliver nucleic acid molecules and achieve a transfection effect, and particularly relates to an ionizable lipid nanoparticle and a preparation method thereof.

Background

Nanoparticle (NPs) based therapeutic and diagnostic systems have been extensively studied, with many being approved for clinical use. Currently, an increasing number of NPs with different chemical, physical and biological properties are manufactured with the aim of controlling their fate in the organism and addressing various barriers to transport in vivo.

The process for preparing ionizable lipid vesicles is considered to be simple mixing of water and oil in the conventional art, but it is difficult to ensure that the particle size dispersity is within a reasonable range. At present, most nucleic acid medicines in clinic adopt liposome technology, the safety of the technology is proved, however, high fever rate exists in clinical data, and the high fever rate is probably caused by high dispersion degree of liposome particle size, non-uniform medicine loading and unstable medicine release. In view of the above problems, the present solution provides a technique that can help solve such problems.

Compared with other nucleic acid in-vitro transfection preparations, the ionizable lipid vesicle is more suitable for in-vitro cell transfection under the condition of high uniformity, the delivery efficiency is ensured, the reliability after delivery transfection is ensured, the cytotoxicity of a common transfection reagent is higher, and the liposome is low in toxicity and high in efficiency. The two-step lipid vesicle method can improve the rapid promotion of nucleic acid molecules into animal experiments.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a preparation technology which can be used for testing the in vitro effectiveness of nucleic acid molecules and is suitable for in vivo transfection, simplifies the process from drug screening to animal experiments in drug research and development, provides a lipid vesicle for delivering nucleic acid with higher reliability, improves the delivery efficiency of nucleic acid, and can be used for wide application scenes such as in vitro cells or in vivo transfection and the like.

The technical scheme adopted by the invention is as follows: the invention relates to an ionizable lipid nanoparticle, which is prepared from neutral lipid, ionizable lipid, phospholipid and PEG-lipid.

Preferably, the neutral lipid is cholestrol;

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 and PEG 5000-DMA.

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

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

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

As a further illustrative scheme, the scheme also discloses a preparation method of the ionizable lipid nanoparticle, which comprises the following steps:

s1: preparing raw materials with corresponding molar mass ratio, wherein the raw materials comprise ionizable lipid, phospholipid, neutral lipid and PEG-lipid, and then respectively dissolving the ionizable lipid, the phospholipid, the neutral lipid and the PEG-lipid in an alcohol solution;

s2: mixing the raw materials respectively dissolved in the alcoholic solution to obtain a lipid mixture in the alcoholic solution;

s3: the lipid mixture is added to an aqueous buffer, the volume of which is 3 to 5 times the volume of the lipid mixture, to give final lipid concentrations of 10-33.3% (vol lipid solution/vol aqueous solution) and 0.6-1.8mg/mL (lipid amount divided by total volume after microfluidic mixing), respectively.

Further, microfluidic pre-formed vesicle methods are used in the above steps to generate nanoparticles containing the ionizable lipids.

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

In a preferred scheme, the aqueous buffer solution is 10-100mM phosphate buffer solution, 10-100mM tris (hydroxymethyl) aminomethane buffer solution, or 10-100mM sodium acetate buffer solution, the pH value is 6.3-6.5, and the phosphate buffer solution is preferred.

Further, in the step S3, the ethanol may be volatilized by leaving at room temperature.

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

in a preferred scheme, the stabilized lipid vesicles can be selectively subjected to size classification by adopting hollow fiber tangential flow filtration or a centrifuge, and the particle size dispersion is reduced by purification, so that the particle size is 80-120nm or 180-220nm, the particle size dispersion degree is less than 0.1, and the concentration of PEG on the surface is 20PEG/100nm ² ；

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

Further, the pH of the final lipid vesicle product in an aqueous buffer solution should be similar to the pKa of the ionizable lipid, the neutral ionizable lipid is combined with the phospholipid and the neutral lipid in a lipophilic action manner to form a stable inner core structure, and the PEG of the PEG-lipid forms a hydrophilic layer on the surface to protect the lipid structure, so that thermodynamic equilibrium is achieved.

In the present case, where the nucleic acid molecule is to be encapsulated in such vesicular particles, the mixture of the present case comprises ethanol at a concentration of from 10% (v/v) to 40% (v/v); meanwhile, under the condition of combination of different lipid vesicles, the pH value of the buffer solution can be adjusted to 3-5, so that the buffer solution is suitable for packaging mRNA; in mixing the buffer with ethanol, the ethanol-buffer mixture needs to be warmed to room temperature.

Preferably, the lipid vesicle is added into the ethanol-buffer solution mixed solution, is uniformly mixed, is placed at room temperature for 10-30min, is added with the target nucleic acid solution, is uniformly mixed, and is placed for 10-30min;

the buffer solution is 10mM phosphate buffer solution;

the pH value of the buffer solution is 3-5, and the preferable pH value is 4;

the volume ratio of the ethanol is 10-40%, and the preferred volume ratio of the ethanol is 30%;

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

the mRNA is mCherry and eGFP.

Preferably, the lipid vesicle is loaded with nucleic acid molecules to form a final mature and stable liposome, the mature liposome is injected into a ready-to-use dialysis tube, dialysis and filtration are carried out for 3 hours at low temperature, a phosphate buffer solution with pH of 4 is replaced for 3 times, and 99.9% of ethanol can be removed; the final product can selectively pass through a filter membrane of 0.45 mu m to reduce the particle size dispersion degree, and the final particle size dispersion degree is less than 0.2.

And finally, adding the encapsulated liposome-nucleic acid compound into a dialysis tube, removing the ethanol, or standing at room temperature to volatilize the ethanol, and then storing at the low temperature of 4 ℃.

The ionizable lipid nanoparticle and the preparation method thereof have the beneficial effects that the ionizable lipid nanoparticle is prepared by adopting the scheme:

1. the traditional one-step liposome preparation is divided into two steps, namely firstly preparing lipid 'vesicles', and then carrying drug molecules, wherein the synthesis of the lipid vesicles is simplified lipid composition components, so that the synthesized nanoparticles have better particle size controllability and physical and chemical stability.

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

3. When the liposome vesicle is synthesized, the pH value of the buffer solution is approximate to the pKa of the ionizable lipid, the neutral ionizable lipid, the phospholipid and the neutral lipid are combined in a lipophilic force action mode to form a stable inner core structure, and PEG of the PEG-lipid forms a hydrophilic layer on the surface to protect the lipid structure, so that thermodynamic balance is achieved, and high-uniformity particle size dispersion can be quickly achieved; even after the lipid vesicles having high uniformity of particle size carry nucleic acid molecules, the dispersion degree of particle size is kept at 0.2 or less. The nucleic acid transfection based on this is more reliable.

4. Compared with the traditional film method and the ultrasonic method, the micro-fluidic method is adopted for preparing the lipid vesicles, the dispersion degree of the particle size can be controlled to be below 0.1, the particle size can be kept below 0.2 after mRNA is loaded in the later period, and the preparation method meets international important parameter indexes such as FDA (food and drug administration).

5. Compared with the traditional liposome, the lipid vesicle prepared by the method has higher in-vivo and in-vitro transfection property after carrying mRNA, can actively target the liver or actively not target the liver, and has the effect of improving the treatment window in the field of specific diseases.

6. The nucleic acid-carrying method is a method of lowering the pH to positively charge the ionizable lipid and thereby attracting and encapsulating the negatively charged nucleic acid molecule. The second solution ethanol in the product can accelerate the fluidity of lipid molecules in formed vesicles, and better entrap attracted nucleic acid molecules into nano bodies. The purpose of removing the ethanol is to make the lipid or lipid-nucleic acid complex more coagulated, improving the stability of the product.

7. Except for low toxicity of the liposome, the product can be mixed with specific mRNA before administration according to actual application requirements, and other operations such as preparing nano particles by compounding with the mRNA in advance are not needed, so that the application is more flexible, and most importantly, the particle size is accurately controlled, so that the reliability of experimental data is enhanced.

8. Secondly, the high toxicity of the traditional infectious agents is solved, the traditional infectious agents cannot be directly applied to animal experiments, and meanwhile, the reliability of transfection from in vitro cells to in vivo cells is improved.

Drawings

FIG. 1 shows the lipid nanoparticle size under standard synthetic conditions in this protocol;

FIG. 2 is a graph showing an example of comparing the cell transfection efficiency of lipo in vitro at the same concentration after loading the nucleic acid molecule of eGFP in the liposome vesicle of example 1;

FIG. 3 is a graph showing an example of comparing the efficiency of cell transfection of lipo in vitro at the same concentration after loading a mcerry nucleic acid molecule in the liposome vesicle in example 2;

fig. 4 is an exemplary diagram of example 3 using lipid vesicles to encapsulate Cas9 mRNA and short-chain grnas, comparing Cas 9-encapsulating proteins plus grnas;

FIG. 5 is a mCherry nucleic acid sequence;

FIG. 6 is an eGFP nucleic acid sequence.

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As shown in FIGS. 1-6, the ionizable lipid nanoparticle of the invention comprises neutral lipid, ionizable lipid, phospholipid, and PEG-lipid.

Preferably, the neutral lipid is cholestrol;

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 and PEG5000-DMA, preferably PEG2000-DMG.

The scheme also discloses a preparation method of the ionizable lipid nanoparticle, which comprises the following steps:

s1: preparing raw materials with corresponding molar mass ratio, wherein the raw materials comprise 50% of ionizable lipid, 10% of phospholipid, 38% of neutral lipid and 2% of PEG-lipid, and then respectively dissolving the ionizable lipid, the phospholipid, the neutral lipid and the PEG-lipid in 97% of ethanol;

s2: mixing the above materials respectively dissolved in 97% ethanol to obtain lipid mixture in ethanol;

s3: adding a lipid mixture into 10mM phosphate buffer solution with a pH value of 6.5, and mixing the lipid mixture and an aqueous buffer solution by a microfluidic method, wherein the microfluidic human-shaped micron Y-shaped channel is provided with phosphate buffer solution and ethanol-lipid mixture injected into two ends simultaneously, and the microfluidic injection rate is 3mL/min, wherein the microfluidic chip is 60-120 μm high, 100-200 μm wide, and is distributed with 20-31 μm high and 30-50 μm thick herringbone grid baffles, wherein the volume of the aqueous buffer solution is 3-5 times of the lipid mixture (10-33.3% of lipid solution/vol aqueous solution), and the volume of the aqueous buffer solution is 3-5 times of the lipid mixture, so as to respectively obtain final lipid concentrations of 10-33.3% (vol lipid solution/vol aqueous solution) and 0.6-1.8mg/mL (lipid amount divided by the total volume after microfluidic mixing).

The obtained lipid vesicles are injected into a ready-to-use dialysis tube, preferably with a 25k filter pore size, dialyzed at 4 ℃ for 24h, and the buffer is exchanged 3-5 times, preferably with a buffer of 10mM phosphate buffer, pH 6.5. And (5) obtaining the final lipid vesicle reagent I product after dialysis.

The product also includes a reagent number two, preferably for mixing the product number one with the target nucleic acid molecule. Reagent two is preferably 20mM phosphate buffer, pH 3, containing preferably 20% by volume of ethanol.

The function of this product is to efficiently carry an mRNA molecule, preferably mCherry or eGFP (sequences shown in FIGS. 5 and 6), into a liposome vesicle in reagent No. one. The specific operation is to mix the reagent of the first product in the reagent of the second buffer solution at room temperature and to stand for 10-30min. Then adding the target nucleic acid molecules into the reagent, uniformly mixing, and standing for 10-30min, so that more than 95% of nucleic acid molecules can be successfully carried. The preferred mass ratio of nucleic acid to liposome vesicle is 1.

And finally, adding the encapsulated lipid vesicle-nucleic acid complex (namely the mature liposome) into a dialysis tube to remove the ethanol, or standing at room temperature to volatilize the ethanol, and then storing at a low temperature of 4 ℃.

As shown in fig. 1, the particle size of the lipid vesicle under standard synthesis conditions is shown. The figure shows the particle size and particle size dispersion exhibited by 9 different compositions and ratios of lipids at different synthesis rates. 9 are each independently

Ionizable lipids: neutral lipid: the ratio of PEG-lipid is 50 percent to 45 percent to 5 percent, 60 percent to 35 percent to 5 percent, 70 percent to 25 percent to 5 percent,

or an ionizable lipid: phospholipid: neutral lipid: the ratio of PEG-lipid is 50%:10%: 38.5%: 1.5%.

50％∶10％∶39％∶1％，50％∶10％∶39.5％∶0.5％，

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

Example 1, as shown in figure 2, ionizable lipids: a phospholipid: neutral lipid: the PEG-lipid ratios ranged from 50%:10%: 38.5%: 1.5%, and the liposome vesicles loaded with eGFP nucleic acid molecules at a microfluidic injection rate of 3mL/min, compared to lipofectamine (lipo) transfection efficiency in vitro at the same concentration. The liposome vesicle shows higher transfection efficiency, lipo shows certain cytotoxicity at the concentration, and the cells adopt HEK395.

Example 2, as shown in figure 3, ionizable lipids: phospholipid: neutral lipid: the PEG-lipid ratio ranges from 50%:10%: 38.5%: 1.5%, the microfluidic injection rate is 3ml per minute, and the liposome vesicles, after carrying mCherry nucleic acid molecules, compare the in vitro cell transfection efficiency of lipo at the same concentration. The liposome vesicles showed higher transfection efficiency, and lipo showed some cytotoxicity. The cells were treated with HEK395.

Example 3, as shown in figure 4, mRNA and short-chain gRNA of Cas9 were encapsulated with lipid vesicles, comparing Cas 9-encapsulating protein plus gRNA. Lipid vesicles showed equally excellent cell editing. The cells were treated with HEK395.

When the lipid vesicle is synthesized, the pH value of the buffer solution is approximate to the pKa of the ionizable lipid, the neutral ionizable lipid, the phospholipid and the neutral lipid are combined in a lipophilic force action mode to form a stable inner core structure, and the PEG of the PEG-lipid forms a hydrophilic layer on the surface to protect the lipid structure, so that thermodynamic balance is achieved, and high-uniformity particle size dispersion can be quickly achieved.

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 are present at a nucleic acid/lipid ratio of about, preferably 3 to 10 wt%. The intermediate mixture may optionally be sized to obtain lipid-encapsulated nucleic acid particles, wherein the lipid fraction is a vesicle preferably having a diameter of 80-120nm, or preferably about 180-220 nm.

The nucleic acid-carrying method is a method of lowering the pH to positively charge the ionizable lipid and thereby attracting and encapsulating the negatively charged nucleic acid molecule. The second solution ethanol in the product can accelerate the fluidity of lipid molecules in formed vesicles and better encapsulate attracted nucleic acid molecules into a nanometer body. The purpose of removing the ethanol is to make the lipid or lipid-nucleic acid complex more coagulated, improving the stability of the product.

In addition to the low toxicity of the liposome, the product can be mixed with specific mRNA before administration according to the actual application requirement, and other operations such as preparing nano particles by compounding with the mRNA in advance and the like are not needed, so that the application is more flexible, and most importantly, the particle size is accurately controlled, so that the reliability of experimental data is enhanced.

Secondly, the transfection efficiency of cells in vitro is much higher than that of normal particle size, i.e. 100nm, with lipid vesicles preferably of particle size 180 to 220 nm. Bringing an indicative direction for the later application of liposomes.

The research and development design of the preparation process of the preparation aims at the problems that the stability of the transfection reagent is poor at the present stage, and the required optimization time is long; secondly, the liposome product has poor stability and high particle size dispersion degree at the present stage. Compared with the existing transfection reagent on the market, the product has more stable performance, is easier to be used as a standard reference substance for in vitro cell effectiveness, and can be directly used for preclinical animal experiments; compared with the same type of nano products in the market, the nano product has the advantages of smaller particle size dispersion degree, smaller toxic and side effects on cells, lower requirement on storage environment and the like; when in use, the gene can be directly matched with a specific mRNA sequence to flexibly meet different application requirements.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. An ionizable lipid nanoparticle, comprising: comprises neutral lipid, ionizable lipid, phospholipid, and PEG-lipid.

2. The ionizable lipid nanoparticle of claim 1, wherein: the neutral lipid is cholestrol;

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 and PEG 5000-DMA.

3. The ionizable lipid nanoparticle of claim 2, wherein: the ionizable lipid is: neutral lipid: the molar ratio of PEG-lipid is 50-90%, 10-50% and 0.1-5%; or an ionizable lipid: phospholipid: neutral lipid: the molar ratio of PEG-lipid is 30-60%, 4-20%, 35-55% and 0.1-5%.

4. The ionizable lipid nanoparticle of claim 3, wherein: the ionizable lipid is: phospholipid: neutral lipid: molar ratio of PEG-lipid 50%:10%:38%:2 percent.

5. A method for preparing ionizable lipid nanoparticles according to any of claims 1-4, comprising the steps of:

s1: preparing raw materials with corresponding molar mass ratio, wherein the raw materials comprise ionizable lipid, phospholipid, neutral lipid and PEG-lipid, and then respectively dissolving the ionizable lipid, the phospholipid, the neutral lipid and the PEG-lipid in an alcohol solution; the alcoholic solution comprises one of methanol, ethanol and acetone, and the volume ratio of the alcoholic solution is 70-99.9%;

s2: mixing the raw materials respectively dissolved in the alcoholic solution to obtain a lipid mixture in the alcoholic solution; the aqueous buffer solution is 10-100mM phosphate buffer solution, 10-100mM tris (hydroxymethyl) aminomethane buffer solution or 10-100mM sodium acetate buffer solution, and the pH value is 6.3-6.5;

s3: adding a lipid mixture into an aqueous buffer solution, and mixing the lipid mixture and the aqueous buffer solution by a microfluidic method, wherein the microfluidic chip is a herringbone grating baffle plate with the height of 60-120 mu m, the width of 100-200 mu m, the height of 20-31 mu m in the middle distribution and the thickness of 30-50 mu m, the microfluidic injection speed is 3-10mL/min, the volume of the aqueous buffer solution is 3-5 times of that of the lipid mixture, so as to respectively obtain 10-33.3% (vol lipid solution/vol aqueous solution) and 0.6-1.8mg/mL of final lipid, and ethanol can be volatilized by standing at room temperature.

6. The method of claim 5, wherein the step of preparing the ionizable lipid nanoparticle comprises: injecting the obtained lipid vesicle into a ready-to-use dialysis tube, dialyzing for 24h at 4 ℃, and replacing the buffer solution for 3-5 times; the stabilized lipid vesicle can be selectively subjected to size classification by hollow fiber tangential flow filtration or a centrifuge, and the particle size dispersion is reduced by purification, so that the particle size is 80-120nm or 180-220nm, the particle size dispersion degree is less than 0.1, and the surface PEG concentration is 20PEG/100nm ² 。

7. The method of claim 6, wherein the step of preparing the ionizable lipid nanoparticle comprises: the pH value of the final lipid vesicle product in an aqueous buffer solution is similar to the pKa of the ionizable lipid, the ionizable lipid with neutral charge is combined with phospholipid and neutral lipid in a lipophilic force action mode to form a stable inner core structure, and PEG of the PEG-lipid forms a hydrophilic layer on the surface to protect the lipid structure, so that thermodynamic equilibrium is achieved.

8. The method of claim 7, wherein the lipid nanoparticle is prepared by the following steps: nucleic acid molecules will be encapsulated into such vesicular particles, and the mixture of this embodiment contains ethanol at a concentration of 10% (v/v) to 40% (v/v); meanwhile, under the condition of combination of different lipid vesicles, the pH value of the buffer solution can be adjusted to 3-5, so that the buffer solution is suitable for packaging mRNA; in mixing the buffer with ethanol, the ethanol-buffer mixture needs to be warmed to room temperature.

9. The method of claim 8, wherein the step of preparing the ionizable lipid nanoparticle comprises: adding the lipid vesicles into the ethanol-buffer solution mixed solution, uniformly mixing, standing at room temperature for 10-30min, adding the target nucleic acid solution, uniformly mixing, and standing for 10-30min;

the buffer solution is 10mM phosphate buffer solution;

the pH value of the buffer solution is 3-5;

the volume ratio of the ethanol is 10-40%;

the mass ratio of the nucleic acid molecules to the lipid is 1: (5-30);

the mRNA is mCherry and eGFP.

10. The method of claim 9, wherein the step of preparing the ionizable lipid nanoparticle comprises: the lipid vesicle is loaded with nucleic acid molecules to form a mature and stable final liposome, the mature liposome is injected into a ready-to-use dialysis tube, dialysis and filtration are carried out for 3 hours at low temperature, a phosphate buffer solution with the pH value of 4 is replaced for 3 times, and 99.9% of ethanol can be removed; the final product can selectively pass through a filter membrane of 0.45 mu m to reduce the particle size dispersion degree, and the final particle size dispersion degree is less than 0.2.

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