Lipid nanoparticle preparation of mRNA (messenger ribonucleic acid) nucleic acid vaccine and preparation method of lipid nanoparticle preparation

Abstract

The invention relates to the field of bioengineering, and discloses an mRNA nucleic acid vaccine lipid nanoparticle preparation, which comprises the following components in percentage by total mole of lipid: dynamic pKa lipid accounting for 15-25% of the total mole of lipid; the phospholipid accounts for 25-35% of the total molar content of the lipid, the invention also discloses a preparation method of the mRNA nucleic acid vaccine lipid nanoparticle preparation, and the preparation method comprises the following steps: S1, dissolving the dynamic pKa lipid, the phospholipid, the cholesterol and the polyethylene glycol modified lipid in ethyl lactate to form an organic phase, and the total concentration of the lipid is 5-15mg / mL. By designing the dynamic pKa lipid and optimizing the preparation process, not only can the encapsulation efficiency and delivery efficiency of mRNA be remarkably improved, but also a green solvent can be used for replacing a traditional solvent, solvent residues and environmental burden are reduced, in addition, by improving the microfluidic technology, the production efficiency and consistency of LNP are improved, and the application prospect is wide. The problems of stability and consistency in a traditional LNP preparation process are solved.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to an mRNA nucleic acid vaccine lipid nanoparticle preparation and a preparation method thereof. Background Art

[0002] In recent years, mRNA technology, as a new gene therapy and vaccine platform, has made significant progress in infectious disease vaccines (such as the new coronavirus vaccine) and gene therapy. mRNA vaccines induce the body to produce an immune response by delivering mRNA encoding the target antigen into cells. However, the delivery of mRNA still faces many challenges. Since the mRNA molecule itself is unstable and easily degraded, and it is difficult for it to enter the target cells efficiently, how to effectively deliver mRNA is a major problem in the field of mRNA vaccines and gene therapy.

[0003] As an effective delivery carrier, lipid nanoparticles (LNP) have played a vital role in the research and development of mRNA vaccines. LNP can not only protect mRNA from degradation by the external environment, but also help mRNA enter target cells and promote its release in cells. However, although LNP technology has achieved good application results in mRNA delivery, the existing technology still has the following limitations. First, the encapsulation efficiency of LNP is relatively low, resulting in a large amount of mRNA not being effectively encapsulated, thereby increasing production costs and reducing efficacy. Secondly, the delivery efficiency of LNP is still not ideal in some cases, especially in the acidic environment of the endosomal body, the efficiency of LNP in releasing mRNA is insufficient, which limits its effective delivery in cells. In addition, the traditional LNP preparation process still relies on organic solvents (such as ethanol), which not only increase production costs, but also may bring problems such as solvent residues and environmental pollution.

[0004] Therefore, how to optimize the LNP formulation, improve the encapsulation efficiency and delivery efficiency of mRNA, while reducing production costs and achieving an environmentally friendly preparation process has become a key issue in the current industrialization process of mRNA vaccines and gene therapy drugs.

[0005] In view of the above problems, the present invention provides an mRNA nucleic acid vaccine lipid nanoparticle preparation and a preparation method thereof. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides an mRNA nucleic acid vaccine lipid nanoparticle preparation and a preparation method thereof, which solves the problems of mRNA lipid nanoparticle preparations in terms of encapsulation efficiency, delivery efficiency, production cost and environmental impact.

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: an mRNA nucleic acid vaccine lipid nanoparticle preparation, calculated by total lipid molar percentage, comprises the following components:

[0008] Dynamic pKa lipids, accounting for 15-25% of the total lipid molar content;

[0009] phospholipids, accounting for 25-35% of the total lipid molar content;

[0010] Cholesterol, 40-50% of the total molar content of lipids;

[0011] PEG-modified lipids, accounting for 3-10% of the total lipid molar content;

[0012] The mRNA nucleic acid is encapsulated in the interior of the lipid nanoparticles, wherein the mRNA nucleic acid is bound to the dynamic pKa lipids through electrostatic interaction.

[0013] Preferably, the structure of the dynamic pKa lipid includes a fatty acid chain, an imidazole group and a phosphate linking group, and the dynamic pKa has a protonation ability ranging from 6.5 to 7.5 between an acidic environment and a neutral environment.

[0014] Preferably, the preparation of the dynamic pKa lipid comprises the following specific steps:

[0015] Stearic acid and chlorophosphite are stirred and reacted in dichloromethane to generate a phosphorus oxychloride intermediate;

[0016] reacting the phosphorus oxychloride intermediate with 1-chloro-2,3-propylene oxide under anhydrous conditions to generate a phosphate compound;

[0017] The imidazole group was introduced into the phosphate compound via a nucleophilic substitution reaction to form a dynamic pKa lipid.

[0018] Preferably, the molar ratio of the mRNA to the dynamic pKa lipid is 1:4-1:10.

[0019] A method for preparing an mRNA nucleic acid vaccine lipid nanoparticle preparation is prepared using the component materials of the mRNA nucleic acid vaccine lipid nanoparticle preparation described above, comprising the following steps:

[0020] S1, dissolving dynamic pKa lipids, phospholipids, cholesterol and polyethylene glycol-modified lipids in ethyl lactate to form an organic phase, with a total lipid concentration of 5-15 mg / mL;

[0021] S2, dissolving the mRNA nucleic acid in 50 mM citric acid buffer to form an aqueous phase, with the mRNA concentration being 0.5-2 mg / mL;

[0022] S3, using a microfluidic device to mix the organic phase and the aqueous phase in a volume ratio of 1:3, and during mixing, the flow rates of the organic phase and the aqueous phase are controlled to be 2-10 mL / min and 8-30 mL / min, respectively;

[0023] S4, dialyzing the mixed solution to remove the solvent and unbound mRNA to obtain an mRNA lipid nanoparticle preparation;

[0024] S5. Determine the particle size and polydispersity by dynamic light scattering. Adjust the process parameters to make the particle size 50-150nm and the polydispersity index less than 0.2

[0025] Preferably, in the step S3, the shear force is adjusted by a microfluidic device during the mixing process, and the specific steps are:

[0026] S3.1, control the flow rates of organic phase and aqueous phase to 2-10mL / min and 8-30mL / min respectively, and adjust the flow rate ratio to 1:3;

[0027] S3.2. Ensure particle generation shear forces in the microfluidic mixing zone are in the range of 150-250 dyn / cm 2 ;

[0028] S3.3. Optimize the particle size so that more than 90% of the particles have a diameter within the range of 50-150 nm.

[0029] Preferably, ethyl lactate is used as the organic solvent for dialysis in step S4, and the specific steps are as follows:

[0030] S4.1, dissolving dynamic pKa lipids, phospholipids, cholesterol and polyethylene glycol-modified lipids in ethyl lactate, and controlling the dissolved concentration to 5-15 mg / mL;

[0031] S4.2. Use deionized water for 2-6 hours during the dialysis process, and replace the dialysate every hour;

[0032] S4.3. The residual solvent content is less than 10 ppm as determined by gas chromatography.

[0033] Preferably, the temperature in the step S3 is controlled at 23° C.-27° C. during the mixing process.

[0034] The present invention provides an mRNA nucleic acid vaccine lipid nanoparticle preparation and a preparation method thereof. It has the following beneficial effects:

[0035] 1. The present invention designs a new type of ionized lipid with dynamic pKa regulation function. The present invention can effectively promote the electrostatic interaction between lipid and mRNA in an acidic environment, thereby improving the encapsulation efficiency and intracellular release efficiency of mRNA. The ionized lipids in the prior art, such as DLI n-MC3-DMA, are effective, but their pKa value regulation is not yet precise, which limits the further improvement of the delivery efficiency.

[0036] 2. By introducing specific chemical groups such as imidazole or diamine groups, the dynamic pKa lipids of the present invention can enhance the protonation effect in the acidic environment of the endosomal body, thereby effectively releasing the encapsulated mRNA, significantly improving the delivery efficiency of mRNA, and reducing the dosage requirement of mRNA, thereby reducing the production cost of vaccines or drugs and alleviating potential side effects.

[0037] 3. The present invention significantly improves the efficiency and consistency of the LNP preparation process by optimizing microfluidic technology and green solvent substitution. The existing LNP preparation technology has bottlenecks in process stability and cost control. The present invention can improve production efficiency and ensure particle consistency by adjusting the flow rate, shear force and real-time monitoring module on the microfluidic device. The optimized microfluidic technology can solve the batch difference problem in the existing process and improve the quality control level of LNP preparations.

[0038] 4. The present invention further reduces the risk of solvent residue and environmental pollution by replacing traditional ethanol solvents with green solvents such as ethyl lactate, while meeting the requirements of green pharmaceutical manufacturing and environmental protection. Ethyl lactate has been approved by the FDA for use in pharmaceutical preparations, and its safety and effectiveness have been verified, thereby ensuring the environmental friendliness and sustainability of the preparation process. This improvement not only reduces the cost of waste disposal, but also improves the safety of the preparation, providing a more feasible solution for industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The present invention is a flow chart of the preparation method. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Please refer to the attached Figure 1 The present invention provides an mRNA nucleic acid vaccine lipid nanoparticle preparation, which comprises the following components based on the total lipid molar percentage:

[0042] Dynamic pKa lipids, accounting for 15-25% of the total lipid molar content;

[0043] phospholipids, accounting for 25-35% of the total lipid molar content;

[0044] Cholesterol, 40-50% of the total molar content of lipids;

[0045] PEG-modified lipids, accounting for 3-10% of the total lipid molar content;

[0046] The mRNA nucleic acid is encapsulated in the interior of the lipid nanoparticles, wherein the mRNA nucleic acid is bound to the dynamic pKa lipids through electrostatic interaction.

[0047] Specifically, the dynamic pKa lipid is a key material for mRNA delivery in the present invention, and its uniqueness lies in the ability to adjust its protonation state according to changes in the environmental pH value. In a neutral environment, it can stably bind to mRNA and form a protective barrier through electrostatic action; in an acidic environment (such as endosomes), it can be protonated, destroy the endosomal membrane structure, and promote the release of mRNA. The design of dynamic pKa lipids is to improve the delivery efficiency of mRNA without affecting the stability of the particles.

[0048] Phospholipids (such as DSPC) are the main components of lipid nanoparticle membranes, and their role is to provide structural stability for the particles. Phospholipids enhance the physical strength of the particles by forming a bilayer, while their good biocompatibility ensures the safety of the preparation in the body. The proportion of phospholipids needs to be appropriate. Too much may reduce the fluidity of the particles, while too little may lead to unstable particle structure.

[0049] Cholesterol is an important component of lipid nanoparticles and is used to regulate the fluidity and rigidity of lipid membranes. It can fill the gaps between phospholipid molecules, enhance the stability of the membrane, and prevent the particles from being rapidly degraded in the body. A high proportion of cholesterol can also improve the stability of the particles in the blood and the delivery efficiency.

[0050] By forming an "invisible" protective barrier on the surface of the particles, polyethylene glycol-modified lipids can significantly increase the circulation time of lipid nanoparticles and reduce the possibility of them being cleared by the immune system in the blood. In addition, the hydrophilicity of polyethylene glycol molecules also enhances the dispersibility of particles and prevents particle aggregation.

[0051] mRNA is the functional core material of the preparation of the present invention, which is used to encode the target antigen or therapeutic protein. Since mRNA itself is easily degraded and has poor stability in vivo, it needs to be protected and delivered by lipid nanoparticles to ensure that it can effectively function in cells. The electrostatic interaction between dynamic pKa lipids and mRNA is the basis for achieving efficient encapsulation and delivery.

[0052] The structure of dynamic pKa lipids includes fatty acid chains, imidazole groups and phosphate linking groups, and the dynamic pKa varies in the protonation ability range from 6.5 to 7.5 between acidic and neutral environments.

[0053] Specifically, the fatty acid chains provide the hydrophobic part for the dynamic pKa lipids to be embedded in the bilayer of lipid nanoparticles. This long chain structure ensures that the lipids have good stability in the nanoparticles and can form a dense lipid membrane to protect the encapsulated mRNA from degradation by the external environment.

[0054] The imidazole group is the functional core of the dynamic pKa lipid, which gives it the ability to be dynamically protonated under different pH conditions. In a neutral environment (such as in the blood), the imidazole group remains in an incompletely protonated state, which helps maintain the stability of the particles; while in an acidic environment (such as in the endosome), the imidazole group is rapidly protonated, destroying the endosomal membrane structure and promoting the release of mRNA. This feature significantly improves the delivery efficiency of mRNA.

[0055] The phosphate linker is an important bridge that connects the imidazole group to the fatty acid chain, and at the same time gives the dynamic pKa lipid a certain biodegradability, ensuring its safe metabolism in the body. The introduction of phosphate also enhances the hydrophilicity of the lipid, which helps to improve the dispersibility and encapsulation efficiency of the lipid molecule.

[0056] The pKa value of dynamic pKa lipids is designed to be in the range of 6.5-7.5, which just covers the range of changes between the endosomal acidic environment (pH ~ 5.5) and the extracellular neutral environment (pH ~ 7.4). This design enables lipids to exhibit different functions in the following two environments:

[0057] Neutral environment: Dynamic pKa lipids mainly maintain a neutral state, forming a stable electrostatic complex with mRNA to protect mRNA from degradation.

[0058] Acidic environment: Under acidic conditions, the imidazole group is rapidly protonated, destroying the stability of the endosomal membrane, triggering the release of mRNA, and ensuring its effective expression in the cell.

[0059] Improve intracellular release efficiency: The protonation properties of dynamic pKa lipids in acidic environments enable them to efficiently release mRNA, thereby improving delivery efficiency.

[0060] Optimizing encapsulation performance: The electrostatic effect of the imidazole group and the hydrophobicity of the fatty acid chain work synergistically to ensure efficient encapsulation of mRNA.

[0061] Provide stability and safety: The phosphate linking group makes the lipid biodegradable, ensuring that it can be metabolized in the body after completing the delivery mission, reducing the risk of toxicity.

[0062] Through the coordinated design of fatty acid chains, imidazole groups and phosphate linking groups, dynamic pKa lipids achieve dynamic switching of functions under neutral and acidic environments, meeting the multiple requirements of the mRNA delivery system for protection, release and biocompatibility, and are the core innovation of the present invention to improve delivery efficiency and safety.

[0063] The preparation of dynamic pKa lipids includes the following specific steps:

[0064] Stearic acid and chlorophosphite are stirred and reacted in dichloromethane to generate a phosphorus oxychloride intermediate;

[0065] reacting the phosphorus oxychloride intermediate with 1-chloro-2,3-propylene oxide under anhydrous conditions to generate a phosphate compound;

[0066] The imidazole group was introduced into the phosphate compound via a nucleophilic substitution reaction to form a dynamic pKa lipid.

[0067] The molar ratio of mRNA to dynamic pKa lipid was 1:4-1:10.

[0068] Specifically, in the mRNA lipid nanoparticle preparation, the main function of the dynamic pKa lipid is to bind to mRNA through electrostatic interaction to form a stable nanostructure. mRNA is a macromolecule with a strong negative charge, and the protonation property of the dynamic pKa lipid provides a positive charge to form a stable complex with mRNA. The molar ratio range is set to 1:4-1:10 to ensure that the dynamic pKa lipid can cover all negatively charged sites of mRNA and achieve efficient electrical neutralization, thereby improving the encapsulation efficiency of mRNA and particle stability.

[0069] The molar ratio of dynamic pKa lipids to mRNA affects the release performance in the endosomal environment. When the molar ratio is too low (<1:4), the positive charge of the dynamic pKa lipid is insufficient to effectively bind and protect mRNA, resulting in reduced encapsulation efficiency; when the molar ratio is too high (>1:10), too much dynamic pKa lipid may cause electrical instability on the particle surface, affecting the adsorption and endocytosis efficiency of the cell membrane. Therefore, the molar ratio design of 1:4-1:10 can strike a balance between protecting mRNA and delivery efficiency.

[0070] Dynamic pKa lipids have strong release capacity in cells, but excessive use may increase potential cytotoxicity. By controlling the molar ratio within the range of 1:4-1:10, it can provide sufficient charge neutralization capacity to ensure the stability and release efficiency of mRNA, while avoiding damage to the cell membrane by excessive lipids, ensuring the safety of the delivery system.

[0071] The design of the molar ratio range also needs to consider feasibility in industrial production. The ratio of 1:4-1:10 is easy to operate under both laboratory and industrial conditions, and can achieve efficient use of raw materials and reduce production waste. At the same time, this range is perfectly compatible with the existing LNP preparation process, avoiding process fluctuations caused by imbalanced ratios.

[0072] A method for preparing an mRNA nucleic acid vaccine lipid nanoparticle preparation is prepared using component materials of an mRNA nucleic acid vaccine lipid nanoparticle preparation, comprising the following steps:

[0073] S1, dissolving dynamic pKa lipids, phospholipids, cholesterol and polyethylene glycol-modified lipids in ethyl lactate to form an organic phase, with a total lipid concentration of 5-15 mg / mL;

[0074] S2, dissolving the mRNA nucleic acid in 50 mM citric acid buffer to form an aqueous phase, with the mRNA concentration being 0.5-2 mg / mL;

[0075] S3, using a microfluidic device to mix the organic phase and the aqueous phase in a volume ratio of 1:3, and during mixing, the flow rates of the organic phase and the aqueous phase are controlled to be 2-10 mL / min and 8-30 mL / min, respectively;

[0076] S4, dialyzing the mixed solution to remove the solvent and unbound mRNA to obtain an mRNA lipid nanoparticle preparation;

[0077] S5. Determine the particle size and polydispersity by dynamic light scattering, and adjust the process parameters to make the particle size 50-150nm and the polydispersity index less than 0.2.

[0078] In step S3, the shear force is adjusted by a microfluidic device during the mixing process, and the specific steps are as follows:

[0079] S3.1, control the flow rates of organic phase and aqueous phase to 2-10mL / min and 8-30mL / min respectively, and adjust the flow rate ratio to 1:3;

[0080] S3.2. Ensure particle generation shear forces in the microfluidic mixing zone are in the range of 150-250 dyn / cm 2 ;

[0081] S3.3. Optimize the particle size so that more than 90% of the particles have a diameter within the range of 50-150 nm.

[0082] In step S4, ethyl lactate is used as the organic solvent for dialysis, and the specific steps are as follows:

[0083] S4.1, dissolving dynamic pKa lipids, phospholipids, cholesterol and polyethylene glycol-modified lipids in ethyl lactate, and controlling the dissolved concentration to 5-15 mg / mL;

[0084] S4.2. Use deionized water for 2-6 hours during the dialysis process, and replace the dialysate every hour;

[0085] S4.3. The residual solvent content is less than 10 ppm as determined by gas chromatography.

[0086] The temperature in step S3 is controlled at 23°C-27°C during the mixing process.

[0087] Embodiment 1:

[0088] Ingredients and recipe:

[0089] Dynamic pKa lipid: 20 mol%;

[0090] Phospholipids (DSPC): 30 mol%;

[0091] Cholesterol: 45 mol%;

[0092] Polyethylene glycol modified lipid (DSPE-polyethylene glycol 2000): 5 mol%;

[0093] mRNA nucleic acid concentration: 1 mg / mL (encoding green fluorescent protein, eGFP).

[0094] Preparation steps:

[0095] Organic phase preparation: Dynamic pKa lipids, phospholipids, cholesterol and polyethylene glycol-modified lipids were dissolved in ethyl lactate with a total lipid concentration of 10 mg / mL and stirred for 30 min to form a uniform lipid organic phase.

[0096] Preparation of aqueous phase: mRNA nucleic acid was dissolved in 50 mM citric acid buffer, pH 4.5, to form an aqueous phase.

[0097] Microfluidic mixing: Use microfluidic equipment to mix the organic phase and the aqueous phase in a volume ratio of 1:3. The organic phase flow rate is 5 mL / min, the aqueous phase flow rate is 15 mL / min, and the shear force is adjusted to 200 dyn / cm 2 .

[0098] Dialysis purification: Place the mixed solution in a dialysis bag and dialyze with deionized water for 4 hours, replacing the dialysate every hour to ensure that the residual solvent ethyl lactate is less than 10 ppm.

[0099] Characterization: Dynamic light scattering was used to determine the particle size (80 nm) and polydispersity index (PDI of 0.18) of the nanoparticles.

[0100] Summary: Example 1 verifies the feasibility of the basic preparation process in the technical solution and successfully prepares mRNA lipid nanoparticle preparations with a particle size of 80 nm and a polydispersity index of 0.18. By controlling the proportion of dynamic pKa lipids to 20 mol%, using traditional microfluidics and optimizing the shear force to 200 dyn / cm 2 , achieving high mRNA encapsulation efficiency. Using ethyl lactate as an organic solvent combined with a dialysis purification process effectively reduced the amount of residual solvent to less than 10ppm, meeting the requirements of safety and stability.

[0101] The basic process of this example provides a reliable reference for subsequent optimization, proves the key role of dynamic pKa lipids in mRNA delivery and the stability of the basic process, and lays the foundation for further optimization of the technical solution.

[0102] Embodiment 2:

[0103] Ingredients and recipe:

[0104] Dynamic pKa lipid: 25 mol%;

[0105] Phospholipids (DSPC): 25 mol%;

[0106] Cholesterol: 45 mol%;

[0107] Polyethylene glycol modified lipid (DSPE-polyethylene glycol 2000): 5 mol%;

[0108] mRNA nucleic acid concentration: 0.8 mg / mL (encoding green fluorescent protein, eGFP).

[0109] Preparation steps:

[0110] Organic phase preparation: Dynamic pKa lipids, phospholipids, cholesterol and polyethylene glycol-modified lipids were dissolved in ethyl lactate with a total lipid concentration of 12 mg / mL and stirred for 30 min to form a uniform lipid organic phase.

[0111] Preparation of aqueous phase: mRNA nucleic acid was dissolved in 50 mM citric acid buffer, pH 4.8, to form an aqueous phase.

[0112] Microfluidic mixing: Use microfluidic equipment to mix the organic phase and the aqueous phase in a volume ratio of 1:3. The organic phase flow rate is 6mL / min, the aqueous phase flow rate is 18mL / min, and the shear force is adjusted to 220dyn / cm 2 .

[0113] Dialysis purification: Place the mixed solution in a dialysis bag and dialyze with deionized water for 6 hours, replacing the dialysate every hour to ensure that the residual solvent ethyl lactate is less than 8 ppm.

[0114] Characterization: Dynamic light scattering was used to determine the particle size (75 nm) and polydispersity index (PDI of 0.15) of the nanoparticles.

[0115] Summary: Example 2 optimizes the proportion of dynamic pKa lipids based on the basic process, increasing it from 20 mol% to 25 mol%. By adjusting the lipid formula and pH value (4.8), the mRNA encapsulation efficiency (93%) was significantly improved, the particle size (75 nm) and PDI (0.15) were reduced, and high uniformity of the particles was achieved. At the same time, the dialysis time was appropriately extended to 6 h, which effectively reduced the amount of residual solvent (<8 ppm), further improving the safety of the preparation.

[0116] This example demonstrates the central role of the dynamic pKa lipid ratio in particle structure and performance, and provides a feasible path for optimizing the physicochemical properties of mRNA lipid nanoparticles, reflecting the flexibility and advancement of the technical solution.

[0117] Embodiment three:

[0118] Ingredients and recipe:

[0119] Dynamic pKa lipid: 20 mol%;

[0120] Phospholipids (DSPC): 30 mol%;

[0121] Cholesterol: 40 mol%;

[0122] Polyethylene glycol modified lipid (DSPE-polyethylene glycol 2000): 10 mol%;

[0123] mRNA nucleic acid concentration: 1.2 mg / mL (encoding green fluorescent protein, eGFP).

[0124] Preparation steps:

[0125] Organic phase preparation: Dynamic pKa lipids, phospholipids, cholesterol and polyethylene glycol-modified lipids were dissolved in ethyl lactate with a total lipid concentration of 10 mg / mL and stirred for 30 min to form a uniform lipid organic phase.

[0126] Preparation of aqueous phase: mRNA nucleic acid was dissolved in 50 mM citric acid buffer, pH 4.5, to form an aqueous phase.

[0127] Microfluidic mixing: Use microfluidic equipment to mix the organic phase and the aqueous phase in a volume ratio of 1:3. The organic phase flow rate is 8mL / min, the aqueous phase flow rate is 24mL / min, the mixing temperature is controlled at 25℃, and the shear force is adjusted to 230dyn / cm 2 .

[0128] Dialysis purification: Place the mixed solution in a dialysis bag and dialyze with deionized water for 4 hours, replacing the dialysate every hour to ensure that the residual solvent ethyl lactate is less than 6 ppm.

[0129] Characterization: Dynamic light scattering was used to determine the particle size (70 nm) and polydispersity index (PDI of 0.12) of the nanoparticles.

[0130] Summary: Example 3, based on the optimization of basic process and dynamic pKa lipid ratio, combined with temperature control (mixing temperature is 25°C), further improved the quality and performance of the preparation. By increasing the proportion of polyethylene glycol-modified lipids to 10 mol% in the formula, the stability of the particles was enhanced and the lipid phase separation problem was effectively avoided. The particle size of the optimized preparation was reduced to 70nm, the PDI was reduced to 0.12, and the encapsulation efficiency was increased to 95%. In addition, by controlling the shear force (230 dyn / cm 2 ) and strict dialysis process (residual solvent less than 6ppm), significantly improving the safety and uniformity of the preparation.

[0131] This example demonstrates the remarkable effect of combining temperature control technology and ratio fine adjustment in improving formulation stability and delivery efficiency, provides an important reference for the large-scale production and performance optimization of dynamic pKa lipid nanoparticles, and further demonstrates the creativity and practical value of the technical solution.

[0132] Summary:

[0133] Example 1: As a basic process verification, mRNA lipid nanoparticles with uniform size and high encapsulation efficiency were successfully prepared, providing full support for the feasibility of the technical solution.

[0134] Example 2: By optimizing the dynamic pKa lipid ratio and process conditions, the uniformity of the particles and the encapsulation efficiency were further improved, highlighting the important role of ratio control in formulation performance.

[0135] Example 3: Combining temperature control and component optimization significantly improved the stability and delivery performance of the preparation, demonstrating the potential advantages of the technical solution in industrial applications.

[0136] Performance verification of mRNA lipid nanoparticles with different formulations:

[0137] Experimental objective: Three mRNA lipid nanoparticle preparations were prepared by using the preparation methods of Example 1, Example 2 and Example 3, respectively, to verify the effects of different formulations and process conditions on their particle size, encapsulation efficiency (EE), delivery efficiency and stability.

[0138] Experimental materials: dynamic pKa lipids (homemade or commercially synthesized), phospholipids (DSPC), cholesterol (chemically pure), polyethylene glycol-modified lipids (DSPE-polyethylene glycol 2000), mRNA nucleic acid (encoding green fluorescent protein, eGFP), ethyl lactate (analytical grade), 50 mM citrate buffer (pH 4.5 or 4.8), deionized water, phosphate buffer (PBS), phosphite chloride, 1-chloro-2,3-propylene oxide, dichloromethane and stearic acid.

[0139] Experimental steps:

[0140] 1. Nanoparticle Preparation

[0141] Three different mRNA lipid nanoparticle preparations were prepared according to the formulas of Example 1, Example 2 and Example 3. The following are the detailed steps.

[0142] 1.1 Preparation of dynamic pKa lipids

[0143] Reaction materials: stearic acid (C18), chlorophosphite, 1-chloro-2,3-propylene oxide, imidazole.

[0144] step:

[0145] Phosphate reaction: Stearic acid and chlorophosphite are stirred in dichloromethane for reaction. The reaction time is 4 hours and the reaction temperature is room temperature.

[0146] Propylene oxide grafting: The generated phosphorus oxychloride intermediate was reacted with 1-chloro-2,3-propylene oxide under anhydrous conditions for 6 hours at a temperature of 25°C.

[0147] Imidazole compound formation: Phosphate compounds were reacted with imidazole groups to obtain dynamic pKa lipids. The reaction time was 8 h and the reaction temperature was 25 °C.

[0148] Purification: Use column chromatography to separate and collect the target product, and confirm the target molecule using HPLC and mass spectrometry (LC-MS).

[0149] 1.2 Preparation of lipid organic phase

[0150] step:

[0151] Prepare lipid solution: weigh dynamic pKa lipid, phospholipid (DSPC), cholesterol and polyethylene glycol modified lipid (DSPE-polyethylene glycol 2000), and dissolve them in ethyl lactate. The total lipid concentration is 10 mg / mL (Example 1), 12 mg / mL (Example 2), and 10 mg / mL (Example 3).

[0152] Stirring: Stir at 25°C for 30 min to ensure that all lipids are dissolved and a homogeneous lipid organic phase is formed.

[0153] 1.3 Preparation of aqueous phase

[0154] step:

[0155] The mRNA nucleic acid (eGFP) was dissolved in 50 mM citric acid buffer, and the pH values ​​were adjusted to 4.5 (Examples 1 and 3) and 4.8 (Example 2), respectively, and the mRNA concentrations were 1 mg / mL (Examples 1 and 3) and 0.8 mg / mL (Example 2), respectively.

[0156] Make sure the mRNA is completely dissolved and free of clumps.

[0157] 1.4 Microfluidic mixing

[0158] step:

[0159] The organic phase and the aqueous phase were mixed in a microfluidic device at a volume ratio of 1:3.

[0160] The organic phase flow rate was set to 5 mL / min (Example 1), 6 mL / min (Example 2) and 8 mL / min (Example 3), and the aqueous phase flow rate was set to 15 mL / min (Example 1), 18 mL / min (Example 2) and 24 mL / min (Example 3).

[0161] Adjust the shear force to 200 dyn / cm 2 (Example 1), 220dyn / cm 2 (Example 2), 230dyn / cm 2 (Example three).

[0162] When mixing, ensure that the mixing temperature is 25°C to allow the mRNA and lipid to react fully.

[0163] 1.5 Dialysis purification

[0164] step:

[0165] The mixed solution was placed in a dialysis bag (MWCO 10 kDa) and dialyzed in deionized water for 4 h (Examples 1 and 3) or 6 h (Example 2), with the dialysate being replaced every hour.

[0166] After the dialysis, the pH value of the solution was checked to ensure that the residual ethyl lactate was less than 10 ppm (Example 1), 8 ppm (Example 2), and 6 ppm (Example 3).

[0167] 1.6 Nanoparticle Characterization

[0168] Particle size and PD I measurements:

[0169] The particle size distribution of the nanoparticles was analyzed using dynamic light scattering (DLS). The particle size (80 nm for Example 1, 75 nm for Example 2, 70 nm for Example 3) and PDI (0.18 for Example 1, 0.15 for Example 2, 0.12 for Example 3) were recorded.

[0170] Compare the results to the standard to ensure that the nanoparticles are uniform in size and well dispersed.

[0171] 2. Performance Testing

[0172] 2.1 Encapsulation efficiency (EE) determination

[0173] step:

[0174] Unencapsulated mRNA is isolated by ultracentrifugation or column chromatography.

[0175] The encapsulated mRNA concentration was determined using a fluorescence spectrophotometer, and the encapsulation efficiency (EE) was calculated.

[0176] formula:

[0177]

[0178] 2.2 In vitro delivery efficiency

[0179] step:

[0180] HeLa cells were selected and seeded into 6-well plates (5×10 5 cell).

[0181] Three different mRNA lipid nanoparticle formulations (10 μg mRNA per well) were delivered to cells and incubated for 6 h.

[0182] The green fluorescence signal in the cells was detected by flow cytometry and the delivery efficiency was calculated.

[0183] Delivery efficiency formula:

[0184]

[0185] 2.3 Stability test

[0186] step:

[0187] The three preparations were stored at 4°C and room temperature (25°C) for 7 days, respectively.

[0188] Dynamic light scattering (DLS) was used daily to measure particle size changes and PD I changes, and stability during storage was recorded.

[0189] Evaluate residual solvents to ensure they are within specified limits.

[0190] Experimental results analysis:

[0191]

[0192]

[0193] in conclusion:

[0194] Particle size and uniformity: Example 3 has the smallest particle size (70 nm) and the lowest PDI (0.12), indicating that the combination of temperature control technology and formula optimization can significantly improve particle uniformity.

[0195] Encapsulation efficiency: Example 3 had the highest encapsulation efficiency (95%), which verified the binding efficiency of the dynamic pKa lipid with mRNA and the synergistic effect of the polyethylene glycol-modified lipid.

[0196] Delivery efficiency: Example 3 has the highest delivery efficiency (80%), indicating that the optimized formula has significant advantages in intracellular delivery performance.

[0197] Stability: After storage for 7 days, the changes in particle size and PDI of the three preparations were small, proving that the lipid nanoparticles have good storage stability. Example 3 performed best in terms of particle size change and PDI change.

[0198] The process of Example 3 combines temperature control technology and component optimization, showing optimal results in formulation performance, delivery efficiency and stability, further demonstrating the creativity and application potential of the technical solution.

[0199] Performance comparison between traditional preparation technology and the mRNA lipid nanoparticle preparation method of the present invention:

[0200] Experimental objective: By comparing with the traditional lipid nanoparticle preparation method, verify the superiority of the method of the present invention (including Examples 1, 2 and 3) in preparing mRNA lipid nanoparticles in terms of particle size, encapsulation efficiency, delivery efficiency, stability, etc.

[0201] Experimental materials: dynamic pKa lipids (homemade or commercially synthesized), phospholipids (DSPC), cholesterol (chemically pure), polyethylene glycol-modified lipids (DSPE-polyethylene glycol 2000), mRNA nucleic acid (encoding green fluorescent protein, eGFP), ethyl lactate (analytical grade), 50 mM citrate buffer (pH 4.5 or 4.8), deionized water, phosphate buffer (PBS), phosphite chloride, 1-chloro-2,3-propylene oxide, dichloromethane and stearic acid.

[0202] Traditional preparation methods: solvent evaporation or thin film hydration

[0203] Overview of traditional preparation technology:

[0204] Traditional preparation methods usually use thin film hydration method or solvent evaporation method, the steps are as follows:

[0205] The lipids are mixed with an organic solvent (such as chloroform or dichloromethane) to form a thin film.

[0206] After evaporation of the solvent, the film is mixed with an aqueous phase (such as a buffer containing the mRNA) to form nanoparticles.

[0207] Further processing and homogenization were performed by sonication or rotary evaporation.

[0208] Use dialysis to remove residual solvent.

[0209] Disadvantages of traditional preparation methods:

[0210] Non-uniform particle size: Due to the non-uniformity of the solvent evaporation process, the particle size is usually non-uniform.

[0211] Low encapsulation efficiency: Some mRNA nucleic acids may not be effectively encapsulated, resulting in low encapsulation efficiency.

[0212] Poor stability: Lipids are easily denatured during solvent evaporation, affecting the long-term stability of the particles.

[0213] Long preparation time: The solvent evaporation and post-processing process are cumbersome, affecting production efficiency.

[0214] Experimental steps:

[0215] 1. Preparation of three different formulations of mRNA lipid nanoparticles

[0216] Three different formulations of mRNA lipid nanoparticles were prepared according to the methods of Example 1, Example 2 and Example 3. The traditional thin film hydration method was used as a control to prepare mRNA lipid nanoparticles of the same formulation.

[0217] 1.1 Preparation of dynamic pKa lipids (same as in Examples 1 to 3)

[0218] Dynamic pKa lipids were prepared according to the methods of Examples 1, 2 and 3, respectively, to ensure the consistency of lipid components.

[0219] 1.2 Traditional preparation method:

[0220] According to the traditional thin film hydration method steps, dynamic pKa lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids were dissolved in chloroform.

[0221] The solvent was removed by rotary evaporation to form a thin film.

[0222] The film was mixed with mRNA dissolving solution (50 mM citrate buffer, pH 4.5).

[0223] Lipid nanoparticles were formed by sonication, and the solvent was removed by dialysis.

[0224] 2. Performance Characterization

[0225] 2.1 Particle size and polydispersity index (PDI) determination

[0226] Dynamic light scattering (DLS) was used to measure the particle size distribution and PD I value of lipid nanoparticles obtained by each preparation method.

[0227] 2.2 Encapsulation efficiency (EE) determination

[0228] Unencapsulated mRNA was separated by ultracentrifugation or column chromatography, and the concentration of encapsulated mRNA was measured using a fluorescence spectrophotometer to calculate the encapsulation efficiency (EE).

[0229]

[0230] 2.3 In vitro delivery efficiency

[0231] Cell experiments:

[0232] HeLa cells were selected and seeded into 6-well plates (5×10 5 cell).

[0233] mRNA lipid nanoparticles prepared by different preparation methods (10 μg mRNA per well) were delivered to cells and incubated for 6 h.

[0234] Use flow cytometry to detect green fluorescence signals and calculate the delivery efficiency:

[0235]

[0236] 2.4 Stability test

[0237] All prepared lipid nanoparticles were stored at 4°C and room temperature (25°C) for 7 days, and the particle size changes and PD I changes were measured every day using DLS.

[0238] Experimental results:

[0239]

[0240] Result analysis:

[0241] Particle Size and Polydispersity:

[0242] Traditional method: The particle size is 120nm, the PD I is 0.30, the particle size is large and the distribution is uneven, indicating that the traditional method is difficult to ensure the consistency of nanoparticles.

[0243] Example 3: The particle size is the smallest (70 nm) and the PDI is the lowest (0.12), which shows that the method of the present invention has significant advantages in particle size control and dispersibility.

[0244] Encapsulation efficiency (EE):

[0245] Conventional method: The encapsulation efficiency is 70%, which is lower than that of the embodiment of the present invention.

[0246] Example 3: The encapsulation efficiency is the highest (95%), which verifies that the method of the present invention can improve the encapsulation efficiency through the electrostatic interaction between dynamic pKa lipids and mRNA.

[0247] Delivery efficiency:

[0248] Traditional method: The delivery efficiency is 60%, which is lower than that of the method of the present invention.

[0249] Example 3: The delivery efficiency is 80%, indicating that the optimized lipid formulation and process can improve the delivery efficiency of mRNA.

[0250] stability:

[0251] Traditional method: During storage, the particle size changed significantly (+20 nm) and the PDI increased, indicating that the nanoparticles prepared by the traditional method had poor stability.

[0252] Example 3: The particle size changes minimally (+2 nm), and the PDI changes minimally (+0.01), indicating that the lipid nanoparticles prepared by the method of the present invention have better stability.

[0253] Experimental summary:

[0254] Particle size and uniformity: Compared with the traditional preparation method, Example 3 of the present invention shows a smaller particle size and a lower PDI, which proves the superiority of the present invention in controlling the uniformity of lipid nanoparticles.

[0255] Encapsulation efficiency: The method of the present invention improves the mRNA encapsulation efficiency by optimizing the ratio of dynamic pKa lipids, which is significantly higher than the traditional method.

[0256] Delivery efficiency: The delivery efficiency of the method of the present invention is significantly improved, especially in Example 3, which shows an mRNA delivery ability that is superior to that of traditional methods.

[0257] Stability: The nanoparticles prepared by the traditional preparation method have poor stability during storage, while the lipid nanoparticles of the present invention have minimal changes in particle size and PDI after long-term storage, demonstrating their stability and long-term storage advantages.

[0258] In summary, the method of the present invention is superior to the traditional preparation method in terms of particle size control, encapsulation efficiency, delivery efficiency and stability, which proves the superiority and practical application potential of the technology of the present invention.

[0259] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An mRNA nucleic acid vaccine lipid nanoparticle preparation, characterized in that: Calculated by total lipid molar percentage, the composition includes the following components: Dynamic pKa lipids, accounting for 15-25% of the total lipid molar content; phospholipids, accounting for 25-35% of the total lipid molar content; Cholesterol, 40-50% of the total molar content of lipids; PEG-modified lipids, accounting for 3-10% of the total lipid molar content; The mRNA nucleic acid is encapsulated in the interior of the lipid nanoparticles, wherein the mRNA nucleic acid is bound to the dynamic pKa lipids through electrostatic interaction.

2. The mRNA nucleic acid vaccine lipid nanoparticle preparation according to claim 1, characterized in that: The structure of the dynamic pKa lipid includes a fatty acid chain, an imidazole group and a phosphate connecting group, and the dynamic pKa has a protonation ability ranging from 6.5 to 7.5 between an acidic environment and a neutral environment.

3. An mRNA nucleic acid vaccine lipid nanoparticle preparation according to claim 2, characterized in that: The preparation of the dynamic pKa lipid comprises the following specific steps: Stearic acid and chlorophosphite are stirred and reacted in dichloromethane to generate a phosphorus oxychloride intermediate; reacting the phosphorus oxychloride intermediate with 1-chloro-2,3-propylene oxide under anhydrous conditions to generate a phosphate compound; The imidazole group was introduced into the phosphate compound via a nucleophilic substitution reaction to form a dynamic pKa lipid.

4. The mRNA nucleic acid vaccine lipid nanoparticle preparation according to claim 1, characterized in that: The molar ratio of the mRNA to the dynamic pKa lipid is 1:4-1:

10.

5. A method for preparing an mRNA nucleic acid vaccine lipid nanoparticle preparation, characterized in that: The preparation method is based on the component materials of an mRNA nucleic acid vaccine lipid nanoparticle preparation according to any one of claims 1 to 4, comprising the following steps: S1, dissolving dynamic pKa lipids, phospholipids, cholesterol and polyethylene glycol-modified lipids in ethyl lactate to form an organic phase, with a total lipid concentration of 5-15 mg / mL; S2, dissolving the mRNA nucleic acid in 50 mM citric acid buffer to form an aqueous phase, with the mRNA concentration being 0.5-2 mg / mL; S3, using a microfluidic device to mix the organic phase and the aqueous phase in a volume ratio of 1:3, and during mixing, the flow rates of the organic phase and the aqueous phase are controlled to be 2-10 mL / min and 8-30 mL / min, respectively; S4, dialyzing the mixed solution to remove the solvent and unbound mRNA to obtain an mRNA lipid nanoparticle preparation; S5. Determine the particle size and polydispersity by dynamic light scattering, and adjust the process parameters to make the particle size 50-150nm and the polydispersity index less than 0.

2.

6. The method for preparing a mRNA nucleic acid vaccine lipid nanoparticle preparation according to claim 5, characterized in that: In the step S3, the shear force is adjusted by a microfluidic device during the mixing process, and the specific steps are as follows: S3.1, control the flow rates of the organic phase and the aqueous phase to 2-10mL / min and 8-30mL / min respectively, and adjust the flow rate ratio to 1:3; S3.

2. Ensure particle generation shear forces in the microfluidic mixing zone are in the range of 150-250 dyn / cm 2 ; S3.

3. Optimize the particle size so that more than 90% of the particles have a diameter within the range of 50-150 nm.

7. The method for preparing a mRNA nucleic acid vaccine lipid nanoparticle preparation according to claim 5, characterized in that: In the step S4, ethyl lactate is used as the organic solvent for dialysis, and the specific steps are as follows: S4.1, dissolving dynamic pKa lipids, phospholipids, cholesterol and polyethylene glycol-modified lipids in ethyl lactate, and controlling the dissolved concentration to 5-15 mg / mL; S4.

2. Use deionized water for 2-6 hours during the dialysis process, and replace the dialysate every hour; S4.

3. The residual solvent content is less than 10 ppm as determined by gas chromatography.

8. The method for preparing a mRNA nucleic acid vaccine lipid nanoparticle preparation according to claim 5, characterized in that: In the step S3, the temperature is controlled at 23° C.-27° C. during the mixing process.

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