Lipid nanoparticles and uses thereof

Abstract

The application provides a kind of lipid nanoparticle and its application, belong to the field of biological medicine technology.The lipid nanoparticle includes ionizable cationic lipid YK-009, neutral lipid, PEGylated lipid DMG-PEG2000 and cholesterol, and the molar ratio is 30-60:5-20:30-50:0.1-10.The application adopts two-step preparation process, first prepares empty lipid nanoparticle, then encapsulates nucleic acid under weak acidic condition, and then neutralizes in alkaline buffer solution containing sucrose protective agent.Through the synergistic effect of specific lipid formula, two-step process and protective agent, the encapsulation efficiency of nucleic acid lipid nanoparticle remains above 93% after 3 cycles of freeze-thawing from-80 DEG C to room temperature, effectively solving the poor freeze-thawing stability of lipid nanoparticle in the prior art, and being suitable for in vivo and / or in vitro delivery of nucleic acid drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a lipid nanoparticle and its application. Background Technology

[0002] As a core delivery carrier for nucleic acid drugs such as mRNA, the physicochemical stability of lipid nanoparticles directly determines the efficacy and safety of the drugs. Freeze-thaw cycling is a common storage, transportation, and processing operation for lipid nanoparticles. The freeze-thaw process mainly threatens the encapsulation stability of LNPs (lipid nanoparticles) through various physicochemical mechanisms, including mechanical damage from ice crystals, chemical stress caused by freeze concentration, and interfacial denaturation. This impact is not a slight fluctuation, but a serious problem that can lead to decreased encapsulation efficiency, particle aggregation, or even complete failure. Therefore, freeze-thaw stability must be given high priority in the research and development and production of advanced therapies such as mRNA-LNPs, and effective protection strategies must be adopted.

[0003] To address the stability challenges posed by freeze-thaw cycles, researchers in this field generally employ the strategy of adding cryoprotectants. However, existing technologies often use cryoprotectants as independent additives, failing to achieve effective synergy with the formulation and preparation process of LNPs. Furthermore, existing technologies lack systematic research on how the formulation of LNPs affects their freeze-thaw stability; currently, there are no LNP formulations specifically designed for freeze-thaw stability.

[0004] In summary, existing technologies lack a systematic and synergistic design of specific lipid formulations, two-step preparation processes, and protective agents to address freeze-thaw stability issues. Developing lipid nanoparticles that can efficiently and stably prepare high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability is a pressing technical challenge in the field of nucleic acid delivery. Summary of the Invention

[0005] The main objective of this invention is to provide a lipid nanoparticle and its application, in order to solve the problem of poor freeze-thaw stability of lipid nanoparticles in the prior art.

[0006] To achieve the above objectives, according to a first aspect of the present invention, a lipid nanoparticle is provided, the lipid nanoparticle comprising: an ionizable cationic lipid, a neutral lipid, a PEGylated lipid, and cholesterol; wherein the molar ratio of the ionizable cationic lipid, the neutral lipid, the cholesterol, and the PEGylated lipid in the lipid nanoparticle is 30-60:5-20:30-50:0.1-10; the ionizable cationic lipid includes YK-009, the neutral lipid includes distearate phosphatidylcholine, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, or dipalmitoyl phosphatidylcholine, and the PEGylated lipid includes DMG-PEG2000.

[0007] Furthermore, the above-mentioned lipid nanoparticles have a particle size of 50-100 nm and a dispersion index of 0.001-0.2.

[0008] Furthermore, the molar ratio of the above-mentioned ionizable cationic lipids, the above-mentioned neutral lipids, the above-mentioned cholesterol and the above-mentioned PEGylated lipids is 45-48:10-14:39-42:1-3.

[0009] To achieve the above objective, according to a second aspect of the present invention, a method for preparing lipid nanoparticles is provided, comprising dissolving an ionizable cationic lipid, a neutral lipid, a PEGylated lipid, and cholesterol in an alcohol solution to obtain an organic phase, mixing the organic phase with an aqueous phase using a microfluidic device, and then removing the alcohol solution to obtain lipid nanoparticles; wherein the molar ratio of the ionizable cationic lipid, the neutral lipid, the cholesterol, and the PEGylated lipid is 30-60:5-20:30-50:0.1-10; wherein the ionizable cationic lipid includes YK-009, the neutral lipid includes DSPC, and the PEGylated lipid includes DMG-PEG2000.

[0010] Furthermore, the aqueous phase includes a citrate buffer; the pH of the citrate buffer is 4.0 to 4.5; and the flow rate of the microfluidic device is 8 mL / min to 30 mL / min.

[0011] Further, the alcohol solution is removed using a buffer solution; the buffer solution includes Tris-HCl buffer; the pH of the Tris-HCl buffer solution is 3.0~8.0.

[0012] To achieve the above objectives, according to a third aspect of the present invention, a method for preparing a nucleic acid lipid nanoparticle composition is provided, the method comprising: first mixing the above-mentioned lipid nanoparticles or the lipid nanoparticles prepared by the above method with nucleic acid molecules in a weakly acidic solution to obtain nanoparticles containing nucleic acid molecules; and second mixing the above-mentioned nanoparticles containing nucleic acid molecules with an alkaline buffer solution to prepare the above-mentioned nucleic acid lipid nanoparticle composition.

[0013] Further, the weakly acidic solution includes one or more of Tris-HCl buffer, citrate buffer, phosphate buffer, carbonate buffer or acetate buffer; preferably, the nucleic acid molecule includes DNA molecule or RNA molecule.

[0014] Further, in the above-mentioned nucleic acid lipid nanoparticle composition, the molar ratio of nitrogen (N) in the lipid nanoparticles to phosphorus (P) in the nucleic acid molecules is 1-30:1; preferably, the molar ratio of N in the lipid nanoparticles to P in the nucleic acid molecules is 3-10:1; preferably, the above-mentioned alkaline buffer includes a Tris buffer containing sucrose; more preferably, the sucrose content in the above-mentioned Tris buffer containing sucrose is 10-30% (w / v).

[0015] To achieve the above objectives, according to a fourth aspect of the present invention, the use of the above-described lipid nanoparticles, lipid nanoparticles prepared by the above-described method for preparing lipid nanoparticles, or nucleic acid lipid nanoparticle compositions prepared by the above-described method for preparing nucleic acid lipid nanoparticle compositions in in vivo and / or in vitro nucleic acid delivery is provided.

[0016] The present invention provides a lipid nanoparticle comprising ionizable cationic lipids, neutral lipids, PEGylated lipids, and cholesterol. By rationally selecting and scientifically proportioning the specific components of the ionizable cationic lipids, neutral lipids, PEGylated lipids, and cholesterol, a lipid nanoparticle with intact structure, uniform particle size, and good freeze-thaw stability is obtained. This nanoparticle can be effectively used to encapsulate substances such as nucleic acids, which is of great significance for promoting the development of nucleic acid delivery technology. Attached Figure Description

[0017] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0018] Figure 1 The particle size distribution of the nucleic acid lipid nanoparticle solution according to Example 6 of the present invention is shown in a graph at 4°C.

[0019] Figure 2 The encapsulation efficiency of the nucleic acid lipid nanoparticle solution according to Example 6 of the present invention is shown in a graph at 4°C.

[0020] Figure 3 The diagram shows the encapsulation efficiency results of EGFP-LNP solutions prepared by different encapsulation methods according to Example 7 of the present invention.

[0021] Figure 4 The particle size distribution of EGFP-LNP solutions prepared by different encapsulation methods according to Example 7 of the present invention is shown in the figure.

[0022] Figure 5 The figure shows the statistical results of intracellular fluorescence intensity of EGFP under different encapsulation methods according to Example 7 of the present invention.

[0023] Figure 6 The following diagram shows the fluorescence results of intracellular expression fluorescence intensity of EGFP with different encapsulation methods according to Example 7 of the present invention. Detailed Implementation

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0025] As mentioned in the background section, existing lipid nanoparticles suffer from poor freeze-thaw stability. Therefore, in this application, the inventors attempt to provide a lipid nanoparticle comprising ionizable cationic lipids, neutral lipids, PEGylated lipids, and cholesterol. By rationally selecting and scientifically proportioning the specific components of the ionizable cationic lipids, neutral lipids, PEGylated lipids, and cholesterol, a lipid nanoparticle with high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability was obtained. This nanoparticle can be effectively used to encapsulate substances such as nucleic acids, which is of great significance for promoting the development of nucleic acid delivery technology. Therefore, a series of protection schemes are proposed in this application.

[0026] In a first typical embodiment of this application, a lipid nanoparticle is provided, comprising: ionizable cationic lipids, neutral lipids, PEGylated lipids, and cholesterol; wherein the molar ratio of the ionizable cationic lipids, neutral lipids, cholesterol, and PEGylated lipids in the lipid nanoparticle is 30-60 (including but not limited to 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60): 5-20 (including but not limited to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20): 30-50 (including... However, not limited to 30, 31, 32, 33, 34, 35, 36, 37, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 43, 44, 45, 46, 47, 48, 49, or 50): 0.1-10 (including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1). 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10); the above ionizable cationic lipids include YK-009, the above neutral lipids include distearylphosphatidylcholine, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine or dipalmitoylphosphatidylcholine, and the above PEGylated lipids include DMG-PEG2000 (dimyristoylglycerol-polyethylene glycol-2000).

[0027] In a preferred embodiment, the chemical formula of the above-mentioned YK-009 is shown in Formula I below;

[0028] .

[0029] In a preferred embodiment, the molar ratio of the above-mentioned ionizable cationic lipid, the above-mentioned neutral lipid, the above-mentioned cholesterol and the above-mentioned PEGylated lipid is 45-48 (including but not limited to 45, 46, 47 or 48): 10-14 (including but not limited to 10, 11, 12, 13 or 14): 39-42 (including but not limited to 39, 39.5, 40, 40.5, 41, 41.5 or 42): 1-3 (including but not limited to 1, 1.5, 2, 2.5 or 3).

[0030] In a preferred embodiment, the molar ratio of the above-mentioned ionizable cationic lipid, the above-mentioned neutral lipid, the above-mentioned cholesterol and the above-mentioned PEGylated lipid is 45-48 (including but not limited to 45, 46, 47 or 48): 10-14 (including but not limited to 10, 11, 12, 13 or 14): 39.5-41.5 (including but not limited to 39.5, 40, 40.5, 41 or 41.5): 1.5-2.5 (including but not limited to 1.5, 2 or 2.5).

[0031] In a preferred embodiment, the molar ratio of the ionizable cationic lipid, the neutral lipid, the cholesterol, and the PEGylated lipid is 48:10:40:2.

[0032] In this application, the inventors discovered a lipid nanoparticle containing an ionizable cationic lipid, a neutral lipid, a PEGylated lipid, and a cholesterol component. Further, the ionizable cationic lipid includes YK-009, the neutral lipid includes distearylphosphatidylcholine, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, or dipalmitoylphosphatidylcholine, and the PEGylated lipid includes DMG-PEG2000. When the molar ratio of the ionizable cationic lipid, neutral lipid, cholesterol, and PEGylated lipid is, but is not limited to, 30-60:5-20:30-50:0.1-10, the prepared lipid nanoparticle can efficiently and stably encapsulate nucleic acids, exhibiting high transfection efficiency, low cytotoxicity, and good freeze-thaw stability. It can be well used for encapsulating nucleic acids and other substances, which is of great significance for promoting the development of nucleic acid delivery technology.

[0033] Existing technologies lack systematic research on how the composition of LNP formulations affects their freeze-thaw stability. In particular, the effects of different cationic lipids on freeze-thaw stability vary significantly: for example, data from the examples in this application show that the encapsulation efficiency of LNPs using YK-009 as the cationic lipid after freeze-thaw cycles is 94.5%, while the encapsulation efficiency of LNPs using ALC-0315 after freeze-thaw cycles is only 79.8%. Therefore, in the experimental process of this application, the inventors preferably selected YK-009 as the cationic lipid component for preparing LNPs with excellent freeze-thaw stability.

[0034] Cholesterol, a key component of lipid nanoparticles (LNPs), can intercalate into the lipid bilayer, increasing membrane density and mechanical strength, thereby enhancing its resistance to ice crystals and osmotic pressure shocks. The saturation level and phase transition temperature of auxiliary phospholipids (such as DSPC) also affect the membrane's fluidity and stability at low temperatures. Furthermore, while PEG-lipids can provide steric stabilization and prolong in vivo circulation time, conformational changes in the PEG chains during freeze-thaw cycles can also have complex effects. However, the optimal ratio of these lipid nanoparticles to achieve good freeze-thaw stability remains unknown. Therefore, the inventors conducted extensive experiments and, by achieving a molar ratio of ionizable cationic lipids, neutral lipids, cholesterol, and PEGylated lipids of 45-48:10-14:39-42:1-3, prepared lipid nanoparticles with high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability, achieving unexpected technical results.

[0035] The beneficial effects of this invention are reflected in the following three aspects:

[0036] First, the specific lipid formulation lays the material foundation for freeze-thaw stability. The YK-009 cationic lipid selected in this invention, compared to the traditional ALC-0315, better maintains the structural integrity of the LNP during freeze-thaw cycles (encapsulation efficiency after freeze-thaw cycles: 94.5% vs. 79.8%). The DSPC neutral lipid has a higher phase transition temperature (55℃), which enhances the rigidity of the membrane structure at low temperatures. Cholesterol, at a preferred ratio of 39-42%, can insert into the lipid bilayer, increasing the membrane's density and mechanical strength, effectively resisting ice crystals and osmotic pressure shocks. DMG-PEG2000, at a preferred ratio of 1.5-2.5%, provides steric stabilization, preventing LNP aggregation during freeze-thaw cycles. The specific ratio of these four components (48:10:40:2) was optimized through extensive experiments, resulting in good freeze-thaw stability.

[0037] Second, the two-step preparation process provides technical support for the embedding of the protective agent. This invention employs a two-step method of "preparing empty LNPs first, then encapsulating nucleic acids," followed by neutralization in an alkaline buffer (containing sucrose protective agent). This process design allows the sucrose protective agent to effectively embed into the LNP structure, rather than merely remaining on the particle surface. Experiments show that the encapsulation rate of LNPs using this process after freeze-thaw cycles is 94.5%, while the encapsulation rate of LNPs using the traditional one-step method after freeze-thaw cycles is only 12.9% (without protective agent) or 89.3% (with protective agent but different processes), fully demonstrating the importance of the synergistic effect between the two-step process and the protective agent.

[0038] Third, the sucrose protectant forms a multi-layered synergistic protection mechanism with the specific process. The sucrose protectant plays multiple protective roles in this invention: first, as a cryoprotectant, it forms a glassy state during freezing, reducing mechanical damage to the LNP membrane structure from ice crystals; second, it interacts with the phospholipid head groups on the LNP membrane surface through hydrogen bonding, stabilizing the membrane structure and preventing chemical stress damage caused by freeze-thaw concentration; third, it regulates the osmotic pressure of the system, preventing LNP rupture due to osmotic pressure imbalance caused by freeze-thaw concentration; and fourth, it forms a synergistic protection with the PEG chains on the LNP membrane surface, further enhancing the stability of the membrane structure during freeze-thaw cycles. Experimental data show that sucrose concentrations within the range of 10-30% (w / v) provide good freeze-thaw protection, with 15% (w / v) being the best concentration. After three freeze-thaw cycles, the encapsulation rate was 94.5%, nearly eight times higher than the control group (12.1%) without the protectant.

[0039] In summary, this invention achieves a significant improvement in the freeze-thaw stability of nucleic acid lipid nanoparticles through the systematic synergistic design of specific lipid formulations, a two-step preparation process, and sucrose protectants, demonstrating the non-obviousness and inventiveness of the technical solution of this invention.

[0040] In a preferred embodiment, the particle size of the above-mentioned lipid nanoparticles is 50-100 nm (including but not limited to 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm, 90 nm, 91 nm, 92 nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 nm or 100 nm). The dispersion index (PDI) of the above lipid nanoparticles is 0.001-0.2 (0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.2).

[0041] In this application, the dispersion index (PDI) can be used to reflect the degree of dispersion of the particle size distribution of nanoparticles. The higher the value, the wider the size distribution of nanoparticles and the poorer the uniformity. In the field of drug delivery, the PDI value of nanoparticles is usually required to be ≤0.2.

[0042] In this application, the inventors discovered that when the particle size of the above-mentioned lipid nanoparticles is 50-100 nm and the PDI of the above-mentioned lipid nanoparticles is 0.001-0.2, the prepared lipid nanoparticles can efficiently and stably encapsulate nucleic acids, and have the characteristics of high transfection efficiency, low cytotoxicity and good freeze-thaw stability. They can be well used to encapsulate nucleic acids and other substances, which is of great significance for promoting the development of nucleic acid delivery technology.

[0043] In a second typical embodiment of this application, a method for preparing lipid nanoparticles is provided, wherein an organic phase is prepared by dissolving ionizable cationic lipids, neutral lipids, PEGylated lipids and cholesterol in an alcohol solution, and the organic phase is mixed with an aqueous phase by a microfluidic device and the alcohol solution is removed to obtain lipid nanoparticles.

[0044] The molar ratio of the above-mentioned ionizable cationic lipids, the above-mentioned neutral lipids, the above-mentioned cholesterol, and the above-mentioned PEGylated lipids is 30-60 (including but not limited to 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60): 5-20 (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20): 30-50 (including but not limited to 30, 31, ... 32, 33, 34, 35, 36, 37, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 43, 44, 45, 46, 47, 48, 49 or 50): 0.1-10 (including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10); the above ionizable cationic lipids include YK-009, the above neutral lipids include DSPC, and the above PEGylated lipids include DMG-PEG2000.

[0045] In a preferred embodiment, the molar ratio of the above-mentioned ionizable cationic lipid, the above-mentioned neutral lipid, the above-mentioned cholesterol and the above-mentioned PEGylated lipid is 45-48 (including but not limited to 45, 46, 47 or 48): 10-14 (including but not limited to 10, 11, 12, 13 or 14): 39-42 (including but not limited to 39, 39.5, 40, 40.5, 41, 41.5 or 42): 1-3 (including but not limited to 1, 1.5, 2, 2.5 or 3).

[0046] In a preferred embodiment, the molar ratio of the above-mentioned ionizable cationic lipid, the above-mentioned neutral lipid, the above-mentioned cholesterol and the above-mentioned PEGylated lipid is 45-48 (including but not limited to 45, 46, 47 or 48): 10-14 (including but not limited to 10, 11, 12, 13 or 14): 39.5-41.5 (including but not limited to 39.5, 40, 40.5, 41 or 41.5): 1.5-2.5 (including but not limited to 1.5, 2 or 2.5).

[0047] In a preferred embodiment, the molar ratio of the ionizable cationic lipid, the neutral lipid, the cholesterol, and the PEGylated lipid is 48:10:40:2.

[0048] In a preferred embodiment, the aqueous phase comprises a citrate buffer solution; the pH of the citrate buffer solution is 4.0-4.5 (including but not limited to 4, 4.1, 4.2, 4.3, 4.4 or 4.5).

[0049] In a preferred embodiment, the flow rate of the microfluidic device is 8-30 mL / min (including but not limited to 8 mL / min, 9 mL / min, 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, 14 mL / min, 15 mL / min, 16 mL / min, 17 mL / min, 18 mL / min, 19 mL / min, 20 mL / min, 21 mL / min, 22 mL / min, 23 mL / min, 24 mL / min, 25 mL / min, 26 mL / min, 27 mL / min, 28 mL / min, 29 mL / min or 30 mL / min).

[0050] In a preferred embodiment, the alcohol solution is removed using a buffer solution.

[0051] In a preferred embodiment, the buffer solution comprises a Tris-HCl buffer solution; the pH of the Tris-HCl buffer solution is 3-8 (including but not limited to 3, 4, 5, 6, 7 or 8).

[0052] In this application, the inventors discovered that by dissolving ionizable cationic lipids, neutral lipids, PEGylated lipids, and cholesterol in an alcohol solution to prepare an organic phase, and then mixing the organic phase with an aqueous phase using a microfluidic device to remove the alcohol solution, lipid nanoparticles can be obtained that can efficiently and stably encapsulate nucleic acids. These nanoparticles exhibit high transfection efficiency, low cytotoxicity, and good freeze-thaw stability, which is of great significance for promoting the development of nucleic acid delivery technology.

[0053] In a third typical embodiment of this application, a method for preparing a nucleic acid lipid nanoparticle composition is provided. The preparation method includes: mixing the lipid nanoparticles or the lipid nanoparticles prepared by the above method with nucleic acid molecules in a weakly acidic solution to obtain nanoparticles containing nucleic acid molecules; and mixing the nanoparticles containing nucleic acid molecules with an alkaline buffer solution to prepare the nucleic acid lipid nanoparticle composition.

[0054] Currently, the preparation of LNPs mostly employs a one-step method. The specific process involves dissolving lipid components in ethanol to form an organic phase, dispersing nucleic acid molecules in an acidic aqueous solution to obtain an aqueous phase, and using microfluidic technology to achieve rapid mixing of the two phases. Subsequent purification processes, such as dialysis, ultrafiltration, or tangential flow filtration (TFF), are then used, replacing the acidic buffer and ethanol mixture with a neutral physiological buffer. While the one-step method offers high preparation efficiency, it requires precise control of multiple process parameters, including ethanol concentration, pH gradient, and mixing rate. Crucially, when the mRNA sequence changes, the entire preparation process parameters typically need to be re-optimized and adjusted. This technical limitation restricts its potential for rapid response and flexible adaptation in personalized medicine scenarios.

[0055] The two-step method of first preparing empty LNPs and then loading them with drugs is the most commonly used alternative to the traditional one-step method in laboratory screening. A representative commercial product of this technology is the Lipofectamine series of transfection reagents, which are cationic lipid complex systems. However, this system uses cationic lipids that are highly toxic, causing severe damage to sensitive cell lines, and also suffers from low encapsulation efficiency, unstable transfection efficiency, and large, non-uniform nucleic acid-lipid nanoparticles with limited cellular uptake efficiency. Therefore, there is an urgent need to develop a new method for encapsulating nucleic acid molecules with lipid nanoparticles that offer high encapsulation efficiency, stable transfection efficiency, uniform particle size, and high cellular uptake efficiency.

[0056] In this application, the inventors discovered that by first mixing the above-mentioned lipid nanoparticles with nucleic acid molecules in a weakly acidic solution to obtain nanoparticles containing nucleic acid molecules, and then second mixing the above-mentioned nanoparticles containing nucleic acid molecules with an alkaline buffer, the prepared nucleic acid lipid nanoparticle composition has the characteristics of high encapsulation efficiency, high transfection efficiency, low cytotoxicity and good freeze-thaw stability. In this nucleic acid lipid nanoparticle composition, the above-mentioned lipid nanoparticles can effectively encapsulate nucleic acid molecules, which is of great significance for promoting the development of nucleic acid delivery technology.

[0057] In a preferred embodiment, the weakly acidic solution includes one or more of Tris-HCl buffer, citrate buffer, phosphate buffer, carbonate buffer, or acetate buffer.

[0058] In this application, the inventors discovered that when the weakly acidic solution includes, but is not limited to, one or more of Tris-HCl buffer, citrate buffer, phosphate buffer, carbonate buffer, or acetate buffer, the prepared nucleic acid lipid nanoparticle composition has the characteristics of high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability. In this nucleic acid lipid nanoparticle composition, the lipid nanoparticles can effectively encapsulate nucleic acid molecules, which is of great significance for promoting the development of nucleic acid delivery technology.

[0059] In a preferred embodiment, the pH value of the weakly acidic solution is 3-6 (including but not limited to 3, 4, 5 or 6); preferably, the pH value is 4-5.

[0060] In this application, the inventors discovered that when the pH value of the aforementioned weakly acidic solution is between 3 and 6, the prepared nucleic acid lipid nanoparticle composition exhibits high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability. In this nucleic acid lipid nanoparticle composition, the lipid nanoparticles can effectively encapsulate nucleic acid molecules, which is of great significance for promoting the development of nucleic acid delivery technology. In the experimental process of this application, the inventors preferably use a weakly acidic solution with a pH value between 4 and 5 for preparing the nucleic acid lipid nanoparticle composition.

[0061] In a preferred embodiment, the nucleic acid molecule includes a DNA molecule or an RNA molecule; preferably, the RNA molecule includes one or more of mRNA, siRNA, saRNA, pDNA, cirRNA, antisense oligonucleotide (ASO), or non-coding RNA; more preferably, the RNA molecule includes mRNA.

[0062] In this application, the inventors discovered that the aforementioned nucleic acid molecules include DNA or RNA molecules; preferably, when the aforementioned RNA molecules include one or more of mRNA, siRNA, saRNA, pDNA, cirRNA, antisense oligonucleotides (ASO), or non-coding RNA, the preparation of the aforementioned nucleic acid lipid nanoparticle composition can efficiently and stably produce high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability. In the experimental process of this application, the inventors preferably use the aforementioned lipid nanoparticles to encapsulate mRNA nucleic acid molecules.

[0063] In a preferred embodiment, the molar ratio of nitrogen (N) in the lipid nanoparticles to phosphorus (P) in the nucleic acid molecule in the above-mentioned nucleic acid lipid nanoparticle composition is 1-30:1 (including but not limited to 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, etc.). 1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1 or 30:1); preferably, the molar ratio of N element in the lipid nanoparticles to P element in the nucleic acid molecule in the above nucleic acid lipid nanoparticle composition is 3-10:1 (including but not limited to 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1).

[0064] In this application, the molar ratio of nitrogen (N) in lipid nanoparticles to phosphorus (P) in nucleic acid molecules specifically means: in the lipid composition used to encapsulate nucleic acids, the ratio of the total number of moles of nitrogen atoms in protonable cationic lipids (or ionizable lipids) to the total number of moles of phosphorus atoms in the aforementioned nucleic acids, i.e., N / P = (total number of moles of protonable nitrogen atoms) / (total number of moles of phosphorus atoms). The number of moles of nitrogen atoms is calculated based on the amount of ionizable lipids fed, and the number of moles of phosphorus atoms is calculated based on the amount of nucleic acid fed and the average molecular weight of nucleotide residues in the nucleic acid sequence; unless otherwise stated, all N / P ratios in this application are based on the molar ratio of the fed components.

[0065] In this application, the inventors discovered that when the molar ratio of nitrogen (N) to phosphorus (P) in the lipid nanoparticles of the aforementioned nucleic acid lipid nanoparticle composition is 1-30:1, the prepared nucleic acid lipid nanoparticle composition exhibits high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability. In this nucleic acid lipid nanoparticle composition, the lipid nanoparticles can effectively encapsulate nucleic acid molecules, which is of great significance for promoting the development of nucleic acid delivery technology. In the experimental process of this application, the inventors preferably selected a molar ratio of 3-10:1 for the preparation of the nucleic acid lipid nanoparticle composition. Furthermore, in the experimental process of this application, the inventors creatively discovered that although it is generally believed that a high encapsulation efficiency of nanoparticle formulations generally results in a low empty shell rate, when the N / P ratio is unreasonable (including but not limited to 20:1), although the encapsulation efficiency is high, the empty shell rate is still relatively high, failing to meet the requirements for efficient nucleic acid delivery.

[0066] In a preferred embodiment, the first mixing method includes vortex mixing or blow-dry mixing; preferably, the first mixing method includes vortex mixing.

[0067] In this application, the inventors discovered that when the first mixing method includes vortex mixing or blow-dry mixing, the prepared nucleic acid lipid nanoparticle composition exhibits high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability. In this nucleic acid lipid nanoparticle composition, the lipid nanoparticles can effectively encapsulate nucleic acid molecules, which is of great significance for promoting the development of nucleic acid delivery technology. In the experimental process of this application, the inventors preferably selected vortex mixing as the first mixing method to prepare the nucleic acid lipid nanoparticle composition.

[0068] In a preferred embodiment, the alkaline buffer comprises one or more of Tris buffer, phosphate buffer, or glycine-sodium hydroxide buffer; the alkaline buffer comprises Tris buffer.

[0069] In this application, the inventors discovered that when the above-mentioned alkaline buffers include, but are not limited to, one or more of Tris buffer, phosphate buffer, or glycine-sodium hydroxide buffer, the prepared nucleic acid lipid nanoparticle composition exhibits high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability. In this nucleic acid lipid nanoparticle composition, the lipid nanoparticles can effectively encapsulate nucleic acid molecules, which is of great significance for promoting the development of nucleic acid delivery technology. In the experimental process of this application, the inventors preferably used Tris buffer as the alkaline buffer to prepare the nucleic acid lipid nanoparticle composition.

[0070] In a preferred embodiment, the pH of the alkaline buffer solution is 8-10 (including but not limited to 8, 9 or 10), and the mass concentration of the alkaline buffer solution is 5-80 mM (including but not limited to 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM or 80 mM).

[0071] In this application, the inventors discovered that when the pH of the alkaline buffer solution is 8-10 and the mass concentration of the alkaline buffer solution is 5-80 mM, the prepared nucleic acid lipid nanoparticle composition has the characteristics of high encapsulation efficiency, high transfection efficiency, low cytotoxicity and good freeze-thaw stability. In this nucleic acid lipid nanoparticle composition, the lipid nanoparticles can effectively encapsulate nucleic acid molecules, which is of great significance for promoting the development of nucleic acid delivery technology.

[0072] In a preferred embodiment, the alkaline buffer solution further contains a protective agent; preferably, the protective agent includes one or more of sucrose, trehalose, or glycine; more preferably, the protective agent includes sucrose.

[0073] In a preferred embodiment, the sucrose content in the alkaline buffer solution is 10-30% (w / v), including but not limited to 10% (w / v), 11% (w / v), 12% (w / v), 13% (w / v), 14% (w / v), 15% (w / v), 16% (w / v), 17% (w / v), 18% (w / v), 19% (w / v), 20% (w / v), 21% (w / v), 22% (w / v), 23% (w / v), 24% (w / v), 25% (w / v), 26% (w / v), 27% (w / v), 28% (w / v), 29% (w / v), or 30% (w / v).

[0074] Experimental results from embodiments of the present invention demonstrate that the freeze-thaw stability of nucleic acid lipid nanoparticles is significantly improved through a specific lipid formulation (a specific molar ratio of YK-009 cationic lipid, DSPC neutral lipid, cholesterol, and DMG-PEG2000), a two-step preparation process (preparing empty LNPs first, then encapsulating nucleic acids), and the synergistic effect of the protective agent. Experiments show that the nucleic acid lipid nanoparticles prepared by the present invention maintain an encapsulation efficiency of over 94.5% after three freeze-thaw cycles from -80°C to room temperature, which is more than eight times higher than the comparative sample without the protective agent (encapsulation efficiency 12.1%).

[0075] Traditional one-step methods simultaneously mix lipid components with nucleic acids. While the process is simple, it requires precise control of multiple parameters, including ethanol concentration, pH gradient, and mixing rate. Crucially, when the nucleic acid sequence changes, the entire preparation process usually needs to be re-optimized and adjusted. This technical limitation significantly restricts its application potential in personalized medicine. More importantly, LNPs prepared using this one-step method exhibit poor stability during freeze-thaw cycles. Experimental data shows that the encapsulation efficiency can drop to 12.9% after freeze-thaw cycles, severely impacting the product's efficacy and safety.

[0076] While researchers commonly employ cryoprotectants to address freeze-thaw challenges, current techniques often treat them as standalone additives, failing to create effective synergies with the formulation and preparation process of LNPs. For example, directly dialysis to remove the protective solvent after LNP preparation prevents the cryoprotectant from effectively embedding within the LNP structure, resulting in limited improvement in freeze-thaw stability. Experiments show that even with the same lipid formulation, the encapsulation efficiency of LNPs without cryoprotectants after freeze-thaw cycles is only 12.1%, while that with cryoprotectants reaches 94.5%. This significant difference highlights the importance of synergistic design between cryoprotectants and the preparation process.

[0077] In this application, the inventors discovered that the aforementioned alkaline buffer solution also contains a protective agent; preferably, when the protective agent includes one or more of sucrose, trehalose, or glycine, the prepared nucleic acid lipid nanoparticle composition exhibits high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability. In this nucleic acid lipid nanoparticle composition, the lipid nanoparticles can effectively encapsulate nucleic acid molecules, which is of great significance for promoting the development of nucleic acid delivery technology. In the experimental process of this application, the inventors preferably use an alkaline buffer solution containing 10-30% (w / v) sucrose to prepare the nucleic acid lipid nanoparticle composition.

[0078] In a fourth typical embodiment of this application, the application of the above-described lipid nanoparticles, lipid nanoparticles prepared by the above-described method for preparing lipid nanoparticles, or nucleic acid lipid nanoparticle compositions prepared by the above-described method for preparing nucleic acid lipid nanoparticle compositions in in vivo and / or in vitro nucleic acid delivery is provided.

[0079] In this application, the lipid nanoparticles described above, the lipid nanoparticles prepared by the above-described method for preparing lipid nanoparticles, or the nucleic acid lipid nanoparticle compositions prepared by the above-described method for preparing nucleic acid lipid nanoparticle compositions can be well applied in in vivo and / or in vitro nucleic acid delivery processes.

[0080] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0081] Example 1: Preparation of Nucleic Acid Lipid Nanoparticles

[0082] 1) Preparation of empty lipid nanoparticle solution:

[0083] The cationic lipid YK-009 (DMF number: 38665, its compound structure is shown in Formula I below), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, CAS: 816-94-4), cholesterol (chool), and DMG-PEG2000 (1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000) were dissolved in ethanol at a molar ratio of 48:10:40:2 to prepare an organic phase solution with a total lipid concentration of 15 mg / ml. The aqueous phase was prepared as a 50 mM citrate buffer (pH 4.0). The organic and aqueous phases were mixed at a volume ratio of 1:3 using a microfluidic device at a total flow rate of 20 ml / min. After mixing, the solution was dialyzed in Tris-HCl buffer (pH 5.0) to remove ethanol, thus obtaining the LNP solution.

[0084] .

[0085] 2) Nucleic acid import and encapsulation:

[0086] The LNP solution obtained in step 1) was mixed with an aqueous solution containing EGFP (Enhanced Green Fluorescent Protein) mRNA (diluted 1-fold with sterile enzyme-free water) at a volume ratio of 3:1 (the N / P ratio of lipid to nucleic acid was 5). The mixture was vortexed for 20 seconds and then allowed to stand at room temperature for 5 minutes to obtain the mixed solution.

[0087] In this application, the N / P ratio specifically refers to the molar ratio of nitrogen (N) in lipid nanoparticles to phosphorus (P) in nucleic acid molecules, that is, the ratio of the total molar number of nitrogen atoms in protonable cationic lipids (or ionizable lipids) to the total molar number of phosphorus atoms in nucleic acids in the lipid composition used to encapsulate nucleic acids. The molar number of nitrogen atoms is calculated based on the amount of ionizable lipids fed, and the molar number of phosphorus atoms is calculated based on the amount of nucleic acid fed and the average molecular weight of nucleotide residues in the nucleic acid sequence. Unless otherwise stated, all N / P ratios in this application are based on the molar ratio of the fed components.

[0088] 3) Neutralization:

[0089] Add dropwise a Tris buffer solution containing 15% (w / v) sucrose (main components are glycerol, 2-amino-2-(hydroxymethyl)-1,3-propanediol; CAS number 77-86-1) (pH 8.0) to the mixed solution obtained in step 2) for neutralization, and gently shake to mix well to obtain mRNA-LNP (nucleic acid lipid nanoparticle solution).

[0090] Example 2: Quality Detection of Nucleic Acid Lipid Nanoparticles

[0091] 1) Measurement of particle size and PDI:

[0092] The particle size and PDI of the lipid nanoparticles prepared in Example 1 were determined using a nanolaser particle size analyzer (manufacturer: MALVERN; model: ZSU3305) and dynamic light scattering (DLS) method. The refractive index of the dispersant was 1.340, the test temperature was 25℃, and the equilibration time was 30s. The detection results of particle size and PDI are shown in Table 1 below.

[0093] 2) Encapsulation efficiency measurement:

[0094] The encapsulation efficiency of different nucleic acid lipid nanoparticles in the examples was determined according to the instructions of each kit: the encapsulation efficiency of mRNA was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK). The results of the encapsulation efficiency detection are shown in Table 1 below. The “empty lipid nanoparticles” in Table 1 are the LNP solutions prepared according to step 1) in Example 1.

[0095] Table 1

[0096]

[0097] Note: In Table 1, " / " indicates that the item was not measured.

[0098] The experimental results in Table 1 show that the particle size of the empty lipid nanoparticles is about 50 nm, and the particle size increases to about 90 nm after encapsulating mRNA, with an encapsulation efficiency of over 90%.

[0099] 3) Measurement of empty shell ratio:

[0100] Empty LNPs are considered impurities that may reduce vaccine efficacy. The percentage of LNP particles without encapsulated nucleic acids was determined using nanoflow cytometry (NanoFCM) to characterize the empty shell ratio, a key parameter of LNPs. The specific calculation method for the empty shell ratio is shown below.

[0101] .

[0102] The specific operating procedure for detecting the empty shell rate is as follows:

[0103] a) Dilute the sample to a particle concentration of 1×10⁻⁶ 7 ~1×10 9 particles / mL (no aggregation);

[0104] b) RNase digestion pretreatment of LNP samples to be tested to remove non-specifically adsorbed nucleic acids on the particle surface;

[0105] c) Staining was performed using the lipid-penetrating nucleic acid fluorescent dye SYTO 9;

[0106] d) The nanoflow cytometer simultaneously acquires nanoparticle scattering signals and nucleic acid fluorescence signals;

[0107] e) The true empty payload rate of LNP is obtained by statistically analyzing the percentage of fluorescent negative particles among all scattering particles using a scatter plot.

[0108] The experimental results show that:

[0109] Table 2

[0110]

[0111] The experimental results in Table 2 show that the mRNA-LNP samples prepared by this method have a low empty shell rate, indicating that most particles were successfully encapsulated with nucleic acid drugs. The encapsulation process of this composition system is relatively consistent, and the particle structure is more uniform.

[0112] Example 3: Experiment on the formulation composition ratio

[0113] Following the same preparation method as in Example 1, nucleic acid lipid nanoparticles were prepared by adjusting the formulation ratios of cationic lipids, DSPC, and cholesterol. The encapsulation efficiency, particle size, and PDI were measured. The experimental results of nucleic acid lipid nanoparticles with different formulation ratios before and after freeze-thaw are shown in Table 3.

[0114] Table 3

[0115]

[0116] The experimental results in Table 3 show that the encapsulation efficiency of nucleic acid lipid nanoparticles with different formulation ratios was greater than 90% before freeze-thaw cycles. Formulation 3-10 had a larger particle size of 145 nm, while the particle sizes of the other formulations were relatively similar, ranging from 80 to 100 nm. After three freeze-thaw cycles, the particle size and PDI of formulations 3-9 and 3-10 increased significantly, presumably due to the low cholesterol ratio in these two formulations, which led to a decrease in the rigidity of the LNP membrane structure, making it more susceptible to ice crystal damage. Formulation 3-7 also showed a significant increase in particle size and PDI, presumably due to this formulation... The 1% DMG-PEG2000 ratio was too low, causing DMG-PEG2000 to detach from the membrane surface during the freeze-thaw process, resulting in particles losing their protective layer and thus agglomerating. Samples of formulations 3-4, 3-5, and 3-7 showed a PDI greater than 0.3 after freeze-thaw, while samples of formulations 3-1, 3-2, 3-3, and 3-6 (Example 1) showed no significant changes in encapsulation efficiency, particle size, or PDI after freeze-thaw. Considering the results before and after freeze-thaw, formulations 3-1, 3-2, 3-3, and 3-6 exhibited the best formulation stability. Model samples were prepared using the composition ratio of formulation 3-6.

[0117] Example 4: Experiment to investigate the sugar concentration in the neutralization solution

[0118] Following the same preparation method as in Example 1, the samples were neutralized using sucrose solutions of different concentrations in the neutralization step, and the encapsulation efficiency, particle size, and PDI of the nucleic acid lipid nanoparticles were measured. The experimental results of nucleic acid lipid nanoparticles before and after freeze-thaw at different sugar concentrations are shown in Table 4.

[0119] Table 4

[0120]

[0121] The experimental results in Table 4 show that the encapsulation efficiency, particle size, and PDI of nucleic acid lipid nanoparticles prepared with sugar concentrations in the neutralization solution range of 5%–30% were not different before freezing and thawing. After three freeze-thaw cycles, the encapsulation efficiency of the 5% sugar concentration sample decreased significantly, while the particle size and PDI increased significantly, indicating poor freeze-thaw stability. The encapsulation efficiency of the 10%–30% sugar concentration samples decreased slightly after freezing and thawing, while the particle size and PDI increased slightly, indicating good freeze-thaw stability. In summary, the neutralization solution exhibits good freeze-thaw stability within the sugar concentration range of 10%–30%.

[0122] Example 5: Experiment to verify the universality of the method for preparing nucleic acid lipid nanoparticle solutions.

[0123] 1) Comparison of encapsulation effects with different N / P ratios:

[0124] Following the same formulation and method as in Example 1, with the amount of mRNA kept constant, the volume of empty lipid nanoparticle solution added was adjusted to prepare nucleic acid lipid nanoparticles with different N / P ratios. The encapsulation efficiency and empty shell rate were measured. The experimental results of the encapsulation effect of different N / P ratios are shown in Table 5.

[0125] Table 5

[0126]

[0127] In Table 5, " / " indicates that the item was not measured.

[0128] The experimental results in Table 5 show that during the preparation of nucleic acid lipid nanoparticle solutions, the encapsulation effect is poor when the N / P ratio is 1:1; the encapsulation efficiency is greater than 90% when the N / P ratio is 3:1 and 20:1, but the empty shell rate of the sample is higher when the N / P ratio is 20:1. Therefore, the encapsulation effect is better when the N / P ratio is in the range of 3:1 to 10:1.

[0129] 2) Encapsulation effect of different RNA sequence lengths:

[0130] Nucleic acid lipid nanoparticles with different sequence lengths were prepared according to the same formulation ratio and method as in Example 1. In Table 6, the RNA sequence length of sequence 5-2-1 (Example 1) is 1149 nt (sequence shown as SEQ ID NO: 1), the RNA sequence length of sequence 5-2-2 is 1818 nt (sequence shown as SEQ ID NO: 2), and the RNA sequence length of sequence 5-2-3 is 4462 nt (sequence shown as SEQ ID NO: 3). The experimental results of the encapsulation effect of different RNA sequence lengths are shown in Table 6.

[0131] Table 6

[0132]

[0133] The experimental results in Table 6 show that the nanoparticles constructed in Example 1 have a good encapsulation effect on RNA molecules with sequence lengths ranging from 1000 nt to 4000 nt.

[0134] 3) Encapsulation effects of different neutral lipids:

[0135] Nucleic acid lipid nanoparticles were prepared according to the same formulation ratio and method as in Example 1, except that the neutral lipid (DSPC) in step 1) of Example 1 was replaced with the neutral lipids numbered 5-3-1 or 5-3-2 in Table 7. The neutral lipid for nanoparticles numbered 5-3-1 in Table 7 is DOPE (English name: 1,2-dioleoy1 sn-glycero-3-phosphoethanolamine, CAS: 4004-05-1), and the neutral lipid for nanoparticles numbered 5-3-2 in Table 7 is DPPC (English name: 1,2-dipalmitoy1-sn-glycero3-phosphocholine, CAS: 63-89-8). The experimental results of the encapsulation effect of different neutral lipid samples are shown in Table 7.

[0136] Table 7

[0137]

[0138] The experimental results in Table 7 show that the encapsulation efficiency of nucleic acid lipid nanoparticles using DOPE and DPPC as neutral lipids is greater than 90%. The nucleic acid lipid nanoparticle solutions prepared with different neutral lipids all have good encapsulation effects, indicating that the method for preparing nucleic acid lipid nanoparticle solutions has high versatility.

[0139] 4) Encapsulation effects of different nucleic acid types:

[0140] Nucleic acid lipid nanoparticles were prepared according to the same formulation ratio and method as in Example 1. The difference was that the nucleic acid type in nanoparticle 5-4-1 in Table 8 was siRNA (the amino acid sequence of its sense strand is 5′-AGCCCACAGAGUCUACCCAUU-3′ (SEQ ID NO: 4), and the amino acid sequence of its antisense strand is 5′-AUGGGUAGACUCUGUGGGCUU-3′ (SEQ ID NO: 5). The nucleic acid type in nanoparticle 5-4-2 in Table 8 was pDNA (the sequence is shown in SEQ ID NO: 6). The encapsulation efficiency of pDNA was determined using the dsDNA HS kit (Novizan); the encapsulation efficiency of siRNA was determined using the Qubit microRNA Reagent kit. The experimental results of the encapsulation effect of different nucleic acid types are shown in Table 8.

[0141] Table 8

[0142]

[0143] The experimental results in Table 8 show that the composition system of this application has good encapsulation effect on both siRNA and pDNA, and the method for preparing nucleic acid lipid nanoparticle solutions has high versatility.

[0144] 5) Encapsulation effect of empty lipid nanoparticles at different pH values:

[0145] Nucleic acid lipid nanoparticles were prepared according to the same formulation ratio and method as in Example 1, with the only difference being that in the preparation of sample 5-5-1, ethanol was removed by dialyzing in Tris-HCl buffer (pH 3.0) in step 1), and in the preparation of sample 5-5-2, ethanol was removed by dialyzing in Tris-HCl buffer (pH 8.0) in step 1), and the aqueous solution containing EGFP-mRNA was diluted 1-fold with 50 mM citrate buffer in step 2. The experimental results of the encapsulation effect of empty lipid nanoparticles at different pH values ​​are shown in Table 9.

[0146] Table 9

[0147]

[0148] The experimental results in Table 9 show that the empty lipid nanoparticles of this application have good encapsulation effect in the pH range of 3.0-8.0, and the method for preparing nucleic acid lipid nanoparticle solutions has high versatility.

[0149] Example 6 Stability Results

[0150] 1) Freeze-thaw investigation:

[0151] The nucleic acid lipid nanoparticle solution in Example 1 was frozen at -80°C and thawed at room temperature three times to compare the stability before and after freeze-thaw. The results of the freeze-thaw test are shown in Table 10.

[0152] Table 10

[0153]

[0154] Note: In Table 10, " / " indicates that the item was not measured.

[0155] The experimental results in Table 10 show that the encapsulation efficiency, zeta potential and integrity of the mRNA-LNP prepared by the novel encapsulation method did not change significantly before and after freeze-thaw, and the particle size increased by less than 10 nm, still showing good transfection effect. The sample has good freeze-thaw stability.

[0156] 2) Stability of liquid samples:

[0157] The nucleic acid lipid nanoparticle solution prepared using the novel encapsulation method in Example 1 was stored at 4°C in the dark. Samples were taken and analyzed at 0, 1, 7, 15, and 30 days. The particle size results of the nucleic acid lipid nanoparticle solution at 4°C are as follows: Figure 1 As shown, the encapsulation efficiency of the nucleic acid lipid nanoparticle solution at 4℃ is as follows: Figure 2 As shown in Table 11 below:

[0158] Table 11

[0159]

[0160] The experimental results in Table 11 show that the nucleic acid lipid nanoparticles prepared in Example 1 did not show significant changes in encapsulation efficiency and transfection efficiency after being stored at 4°C for 30 days, indicating that the system has good stability.

[0161] 3) Batch-to-batch stability:

[0162] Nucleic acid lipid nanoparticles were prepared according to the same formulation ratio and method as in Example 1. The encapsulation effect of different batches of empty lipid nanoparticle solutions was investigated, and the results of batch-to-batch stability testing are shown in Table 12 below:

[0163] Table 12

[0164]

[0165] The experimental results in Table 12 show that different batches of empty lipid nanoparticles have good encapsulation effects on mRNA, and the system has good stability.

[0166] Example 7 In vitro cell transfection experiment

[0167] 1) Test sample:

[0168] EGFP-LNP solutions prepared using different encapsulation methods.

[0169] The encapsulation efficiency of EGFP-LNP solutions prepared by different encapsulation methods is as follows: Figure 3 As shown, Figure 3 The experimental results show that the encapsulation efficiency of the samples prepared by the re-encapsulation process is no different from that of the traditional one-step method, and both are better than manual encapsulation. Figure 3 In this text, ReCap indicates re-encapsulation, MF indicates microfluidic, and MAN indicates manual encapsulation. The particle sizes of the EGFP-LNP solutions prepared using different encapsulation methods are as follows: Figure 4 As shown, Figure 4 The experimental results show that the particle size of the samples prepared by the re-encapsulation process and the traditional one-step method is less than 100 nm, while the particle size of the manually encapsulated samples is larger, approximately 200 nm. Figure 4 In the text, ReCap stands for Recapping, MF stands for Microfluidics, and MAN stands for Manual Capping.

[0170] The specific method for preparing EGFP-LNP solution using ReCap is the same as in Example 1.

[0171] The specific method for preparing EGFP-LNP solution by MF is as follows: lipid components YK-009, DSPC, chol, and DMG-PEG2000 are dissolved in ethanol at a molar ratio of 48:10:40:2 to form an organic phase. The EGFP stock solution is dissolved in CB buffer (citrate buffer) to obtain an aqueous phase. The two phases are rapidly mixed using microfluidic technology. The solution is then purified by dialysis with neutral Tris buffer (pH=7.4) to remove acidic buffer and ethanol from the system, thus obtaining the solution.

[0172] The specific method for preparing EGFP-LNP solution via MAN is as follows: lipid components YK-009, DSPC, chol, and DMG-PEG2000 are dissolved in ethanol at a molar ratio of 48:10:40:2 to form an organic phase. The EGFP stock solution is dissolved in CB buffer to obtain an aqueous phase. The lipid ethanol phase is rapidly added to the RNA aqueous phase solution, and the mixture is manually mixed by pipetting. The solution is then purified by dialyzing with neutral Tris buffer (pH=7.4) to remove acidic buffer and ethanol from the system, thus obtaining the final product.

[0173] 2) Primary T cell transfection:

[0174] T cell activation: Primary T cells were activated by stimulation with anti-CD3 / CD28 antibody and IL-2 for 48 hours.

[0175] Centrifuge the logarithmically grown activated T cells at 1000 rpm for 3 min and discard the supernatant. Resuspend the cells in serum-free medium to a final volume of 1×10⁻⁶. 6 500 μL of cells were seeded in 24-well plates.

[0176] EGFP-LNP solution was transfected, and different doses (200 ng, 500 ng, 1000 ng) of the test sample were added to each well according to a concentration gradient. The mixture was gently shaken, and a cell control group was also included. Six parallel wells were set up. The cells were incubated at 37°C in a 5% CO2 incubator for 4 hours. After 4 hours, serum-containing complete medium + IL-2 was added, and the cells were cultured further.

[0177] 3) Cell viability assay:

[0178] The cytotoxicity of the nucleic acid lipid nanoparticles in Example 1 was investigated. After 48 h of transfection, the cell plate was removed from the incubator, and 10 μL of LCK-8 solution was added to each well. The plate was mixed by shaking at low speed and then incubated in a 37°C, 5% CO2 incubator for 1 hour. The absorbance at 450 nm was measured using a microplate reader. The cell viability results are shown in Table 13 below.

[0179] Table 13

[0180]

[0181] The experimental results in Table 13 show that, at doses of 200 ng to 1000 ng / well, the cell survival rate in the Example 1 group was >80%. The nucleic acid lipid nanoparticle system of the present invention has low cytotoxicity, but exhibits dose-dependent toxicity characteristics. Subsequently, 500 ng / well was used as a safe and effective dose.

[0182] 4) Expression detection:

[0183] Forty-eight hours after transfection, 500 ng / well samples from the drug-treated group were collected, and EGFP fluorescence was detected using a flow cytometer (LSRFortessa™ X-20). The number and percentage of EGFP-positive cells were counted, and the statistical results of intracellular fluorescence intensity of EGFP expression under different encapsulation methods are as follows: Figure 5 As shown, the fluorescence results of intracellular expression fluorescence intensity of EGFP with different encapsulation methods are as follows: Figure 6 As shown, Figure 5 and Figure 6 The experimental results show that the samples prepared by the re-encapsulation process have high intracellular expression, and the preparation method is stable and reliable. Figure 5 and Figure 6 In the table below, ReCap indicates recapsulation, MF indicates microfluidics, and MAN indicates manual capsulation. The specific experimental results of in vitro cell transfection efficiency are shown in Table 14. The blank control group in Table 14 is the cell control group that was not drugged during transfection.

[0184] Table 14

[0185]

[0186] The experimental results in Table 14 show that the EGFP of the nucleic acid lipid nanoparticles prepared by the novel encapsulation method in Example 1 is... + The average fluorescence intensity is approximately twice that of the one-step method. Both methods have higher transfection efficiency than manual encapsulation, and the preparation method is stable and reliable.

[0187] Example 8: In vivo animal experiments

[0188] 1) Sample preparation:

[0189] Nucleic acid lipid nanoparticles were prepared using the same formulation ratio and method as in Example 1, except that the aqueous phase of nucleic acid was replaced with Fluc mRNA.

[0190] 2) Experimental grouping and treatment plan:

[0191] Experimental animals: 6-8 week old BALB / c mice.

[0192] Grouping and administration: Mice were randomly divided into two groups of 3 each. The drugs were administered via tail vein injection: control group (PBS) and experimental group (Fluc mRNA-LNP, 10 μg mRNA / mouse).

[0193] Twenty-four hours later, mice were anesthetized with isoflurane gas and injected intraperitoneally with 150 mg / kg D-luciferin for in vivo fluorescence imaging (IVIS Lumina XRMS Series III). Images were obtained 10 minutes after administration. Mice were sacrificed after whole-body fluorescence imaging, and in vitro imaging and luciferase activity quantification (RLU / mg tissue) were performed. The results of quantitative fluorescence quantification of isolated mouse organs (% total luminescence intensity and RLU / mg tissue) are shown in Table 15 below.

[0194] Table 15

[0195]

[0196] The experimental results in Table 15 show that the nucleic acid lipid nanoparticles prepared by the method of this application, after intravenous injection, mainly accumulated in the liver (liver targeting), followed by the spleen and lungs. This indicates that the nanodelivery system prepared in this application can achieve good in vivo drug delivery.

[0197] Comparative Example 1: Comparison of different packaging methods and whether or not sucrose is added.

[0198] The process for preparing nucleic acid lipid nanoparticles in this comparative example is basically the same as that in Example 1, except that a traditional one-step method for preparing mRNA-LNP is used, and two groups of samples are set up: one with sucrose and one without. The main specific steps are as follows: lipid components YK-009, DSPC, cholesterol, and DMG-PEG2000 are dissolved in ethanol at a molar ratio of 48:10:40:2 to form an organic phase. EGFP stock solution is dissolved in CB buffer to obtain an aqueous phase. The two phases are rapidly mixed using microfluidic technology, and the system is purified by dialyzing with neutral Tris buffer (pH=7.4) to remove acidic buffer and ethanol. The process in Comparative Example 1 is the same as the MF method in Example 7.

[0199] Comparative Example 2: Comparison of different encapsulation methods

[0200] The process for preparing nucleic acid lipid nanoparticles in this comparative example is basically the same as that in Example 1, except that the lipid ethanol phase is added to the RNA aqueous phase solution, mixed by pipetting, and manually encapsulated. The manual encapsulation method is the same as in Example 7. The process in Comparative Example 2 is the same as the MAN method in Example 7. The mRNA-LNPs prepared according to Comparative Examples 1 and 2 are compared with the mRNA-LNPs obtained in Example 1 in terms of encapsulation efficiency, particle size, and PDI parameters. The experimental results are shown in Table 16 below. The results show that the quality of the mRNA-LNPs prepared by Example 1 is no different from that of the conventional one-step method. The encapsulation efficiency of both methods is higher than that of manual encapsulation. The composition system and preparation method are stable and reliable.

[0201] Comparative Example 3: Necessity of Neutralization

[0202] Nucleic acid lipid nanoparticles were prepared according to the same formulation and method as in Example 1, except that neutralization was not performed after encapsulation. The particle size, PDI, encapsulation efficiency, stability at 4°C for 30 days, and encapsulation efficiency after freeze-thaw cycles of the mRNA-LNP prepared according to the method in Comparative Example 3 were tested. The results are shown in Table 16. The experimental results show that the particle size of the unneutralized sample increased significantly after 30 days at 4°C and after freeze-thaw cycles, and the encapsulation efficiency decreased significantly. At the same time, the acidic system is not suitable for in vitro and in vivo administration, so the sample needs to be neutralized.

[0203] Comparative Example 4: Investigation of Encapsulation with Non-Ionizable Cationic Lipids

[0204] Nucleic acid lipid nanoparticles were prepared according to the same formulation and method as in Example 1, except that the cationic lipid was replaced with DOTAP (1,2-dioleoyl-3-trimethylammonium-propane chloride). The particle size, PDI, cell viability, transfection efficiency, and encapsulation efficiency of the mRNA-LNPs prepared according to the method in Comparative Example 4 were measured. The results are shown in Table 16. The experimental results indicate that, compared with Example 1, the nucleic acid lipid nanoparticles prepared with DOTAP as the cationic lipid had lower in vitro cell transfection efficiency. The YK-009 cationic lipid used in this invention has better delivery performance.

[0205] Comparative Example 5: Investigation into the necessity of adding sugar to the neutralization buffer

[0206] Nucleic acid lipid nanoparticles were prepared according to the same formulation ratio and method as in Example 1. The difference from Example 1 is that sucrose was not added to the neutralization solution. The particle size, PDI, cell viability, transfection efficiency, stability at 4°C for 30 days, and encapsulation efficiency after freeze-thaw cycles of the mRNA-LNP prepared according to the method in Comparative Example 5 were detected. The results are shown in Table 16. The experimental results show that the encapsulation efficiency of the sample decreased significantly after freeze-thaw. Therefore, Tris buffer with added sucrose was selected as the neutralization solution in this application.

[0207] Comparative Example 6: Traditional cationic lipid encapsulation

[0208] Nucleic acid lipid nanoparticles were prepared according to the same formulation ratio and method as in Example 1. The difference from Example 1 is that ALC-0315 ([(4-hydroxybuty)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) was used as the cationic lipid. The particle size, PDI, and encapsulation efficiency after freeze-thaw cycles of the mRNA-LNP prepared according to the method in Comparative Example 6 were detected. The detection results are shown in Table 16. The experimental results show that, compared with Example 1, the sample prepared by ALC-0315 cationic lipid has poor freeze-thaw stability. Therefore, YK-009 was selected to prepare lipid nanoparticles in this invention.

[0209] Experimental Example 1

[0210] The products in Examples 1, 1, 2, 3, 4, 5, and 6 were tested. The methods for detecting particle size, PDI, and encapsulation efficiency were the same as in Example 2. The methods for detecting cell viability were the same as in Example 7. The methods for detecting cell viability and transfection efficiency of nucleic acid lipid nanoparticles at a dose of 500 ng were the same as in Example 6. The methods for detecting transfection efficiency, stability at 4℃ for 30 days, and encapsulation efficiency after freeze-thaw cycles of nucleic acid lipid nanoparticles with different encapsulation methods were the same as in Example 6. The encapsulation efficiency was measured after three freeze-thaw cycles from -80℃ to room temperature. The relevant data are summarized in Table 16.

[0211] Table 16

[0212]

[0213] Note: In Table 16, " / " indicates that the item was not measured.

[0214] The experimental results in Table 16 show that the transfection efficiency of Comparative Examples 2 and 4 was poor, and the acidic system of Comparative Example 3 was not suitable for subsequent cell experiments. Therefore, the nucleic acid lipid nanoparticles prepared under the conditions of specific lipid composition + two-step method + weak acid group + alkaline buffer neutralization (containing protective agent) have better encapsulation of nucleic acids, higher cell viability, higher transfection efficiency, better stability, and higher encapsulation rate after freeze-thaw cycles.

[0215] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0216] This application presents a lipid nanoparticle composed of specific components, exhibiting high encapsulation efficiency, high transfection efficiency, low cytotoxicity, and good freeze-thaw stability. It is well-suited for encapsulating nucleic acids and other substances, enabling targeted nucleic acid delivery. Furthermore, the inventors discovered that by encapsulating nucleic acids and other substances onto the aforementioned lipid nanoparticles using a two-step empty-load encapsulation method, controlling the overall pH value within the 3.0-8.0 range, and neutralizing with a buffer solution containing sucrose as a protective agent, the resulting nucleic acid lipid nanoparticles achieve high encapsulation, high uniformity, high stability, low toxicity, and high transfection efficiency. Moreover, the nucleic acid lipid nanoparticle preparation method provided by this invention is applicable to the encapsulation of various nucleic acid drugs, significantly improving screening efficiency. Furthermore, the two-step preparation method proposed in this invention allows for the scale-up production of empty-load lipid nanoparticles with minimal batch-to-batch particle size variation, stable encapsulation effect, and wide applicability. Furthermore, the nucleic acid lipid nanoparticles prepared by the novel method of this invention showed no significant change in encapsulation efficiency and integrity after three freeze-thaw cycles from -80°C to room temperature; and remained stable after 30 days of storage at 4°C. Finally, the nucleic acid lipid nanoparticles prepared by the method of this invention exhibit good delivery performance both in vivo and in vitro.

[0217] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A lipid nanoparticle, characterized in that, The lipid nanoparticles include: ionizable cationic lipids, neutral lipids, PEGylated lipids, and cholesterol; In the lipid nanoparticles, the molar ratio of the ionizable cationic lipid, the neutral lipid, the cholesterol, and the PEGylated lipid is 30-60: 5-20: 30-50: 0.1-10; The ionizable cationic lipids include YK-009, the neutral lipids include distearate phosphatidylcholine, 1,2-dioleoyl-SN-glycerol-3-phosphatidylethanolamine or dipalmitoyl phosphatidylcholine, and the PEGylated lipids include DMG-PEG2000.

2. The lipid nanoparticles according to claim 1, characterized in that, The lipid nanoparticles have a particle size of 50-100 nm and a dispersion index of 0.001-0.

2.

3. The lipid nanoparticles according to claim 1 or 2, characterized in that, The molar ratio of the ionizable cationic lipid, the neutral lipid, the cholesterol, and the PEGylated lipid is 45-48:10-14:39-42:1-3.

4. A method for preparing lipid nanoparticles, characterized in that, An organic phase was prepared by dissolving ionizable cationic lipids, neutral lipids, PEGylated lipids and cholesterol in an alcohol solution. The organic phase was then mixed with an aqueous phase using a microfluidic device, and the alcohol solution was removed to obtain lipid nanoparticles. The molar ratio of the ionizable cationic lipid, the neutral lipid, the cholesterol, and the PEGylated lipid is 30-60: 5-20: 30-50: 0.1-10; The ionizable cationic lipids include YK-009, the neutral lipids include DSPC, and the PEGylated lipids include DMG-PEG2000.

5. The method according to claim 4, characterized in that, The aqueous phase includes a citrate buffer solution; the pH of the citrate buffer solution is 4.0~4.5; The flow rate of the microfluidic device is 8 mL / min to 30 mL / min.

6. The method according to claim 5, characterized in that, The alcohol solution was removed using a buffer solution; The buffer solution includes a Tris-HCl buffer solution; the pH of the Tris-HCl buffer solution is 3.0 to 8.

0.

7. A method for preparing a nucleic acid lipid nanoparticle composition, characterized in that, The preparation method includes: In a weakly acidic solution, the lipid nanoparticles according to any one of claims 1-3 or the lipid nanoparticles prepared by any one of claims 4-6 are first mixed with nucleic acid molecules to obtain nanoparticles containing nucleic acid molecules. The nucleic acid-containing nanoparticles are mixed with an alkaline buffer solution to prepare the nucleic acid lipid nanoparticle composition.

8. The preparation method according to claim 7, characterized in that, The weakly acidic solution includes one or more of Tris-HCl buffer, citrate buffer, phosphate buffer, carbonate buffer, or acetate buffer. Preferably, the nucleic acid molecule includes a DNA molecule or an RNA molecule.

9. The preparation method according to claim 7, characterized in that, In the nucleic acid lipid nanoparticle composition, the molar ratio of nitrogen (N) to phosphorus (P) in the lipid nanoparticles is 1-30:

1. Preferably, in the nucleic acid lipid nanoparticle composition, the molar ratio of nitrogen (N) in the lipid nanoparticles to phosphorus (P) in the nucleic acid molecule is 3-10:

1. Preferably, the alkaline buffer solution comprises a Tris buffer solution containing sucrose; More preferably, the sucrose content in the Tris buffer containing sucrose is 10-30% (w / v).

10. The use of the lipid nanoparticles according to any one of claims 1-3, the lipid nanoparticles prepared by the method for preparing lipid nanoparticles according to any one of claims 4-6, or the nucleic acid lipid nanoparticle compositions prepared by the method for preparing nucleic acid lipid nanoparticle compositions according to any one of claims 7-9 in in vivo and / or in vitro nucleic acid delivery.

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