Method for storing and using injectable lipid nanoparticles

Abstract

The invention provides a preservation and use method of lipid nanoparticles (LNP) for local injection, and belongs to the technical field of preservation and application of LNP products. The method comprises the following steps: (1) mixing LNP loaded with nucleic acid with a sodium alginate solution, preparing sodium alginate microspheres by adopting a gas shear microfluidic method, and carrying out ionic crosslinking in a calcium ion-containing solution to obtain hydrogel microspheres; (2) preserving the hydrogel microspheres by using a storage solution containing calcium ions, and controlling the concentration of the calcium ions in the storage solution to be 3-5 mM and the pH value to be 6.5-7.0; before local injection, the concentration of calcium ions in the storage liquid is adjusted to be 0.5 mM or 2-5 mM, and the pH value is adjusted to be 7.0-7.5. According to the method, the stability of the LNP in the storage and transportation processes is ensured through hydrogel physical packaging, and meanwhile, the LNP after local injection keeps an efficient nucleic acid delivery function through storage liquid condition adjustment before local injection. The method is low in cost, convenient to operate and suitable for popularization and application of LNP local injection preparations.

Description

Technical Field

[0001] This invention belongs to the field of preservation and application technology of lipid nanoparticle products, and specifically relates to a method for the preservation and use of locally injected lipid nanoparticles. Background Technology

[0002] One of the major challenges in the targeted delivery of bioactive substances is their instability and low cell penetration potential. This is especially true for the delivery of nucleic acid molecules, particularly mRNA. Therefore, proper packaging is crucial for adequate protection and delivery.

[0003] Lipid nanoparticles (LNPs) have been used as important packaging carriers for nucleic acids (mRNA, etc.) to allow delivery into cells and / or intracellular compartments. These LNPs typically contain a mixture of different lipids, such as ionizable lipids, phospholipids, structural lipids (e.g., sterols or cholesterol), PEG lipids, etc.

[0004] Currently, LNP is not only used for systemic delivery via intravenous injection, but also being explored for in-situ injection into local lesions such as joint cavities. Local injection can effectively avoid the uncontrollable liver accumulation problem in systemic delivery, thereby achieving higher bioavailability and safety.

[0005] However, LNPs face numerous challenges during storage. For instance, temperature fluctuations exceeding ±5°C accelerate LNP aggregation, and direct storage requires low temperatures (e.g., -80°C) or frequent freeze-drying, with repeated freeze-thaw cycles easily leading to aggregation. Although materials such as hydrogels have been attempted to encapsulate LNPs, this physical encapsulation results in inefficient release of LNPs after local injection, leading to low cell transfection rates after local delivery.

[0006] Therefore, how to solve the many problems in the storage, transportation and injection application of locally injected LNPs has become an urgent technical problem to be solved. Summary of the Invention

[0007] The present invention aims to solve the aforementioned technical problems by providing a method for the preservation and use of locally injectable lipid nanoparticles. The technical objective of this invention is to address the issues of easy aggregation and instability of lipid nanoparticles during preservation and transportation, as well as the difficulty in maintaining stability and delivery efficiency during injection.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for the preservation and use of locally injected lipid nanoparticles (LNPs), comprising the following steps: (1) Lipid nanoparticles (LNP) were mixed evenly with sodium alginate solution, and then hydrogel microspheres were prepared by gas microfluidics method and collected in calcium chloride solution. (2) Use a preservation solution containing calcium ions to preserve hydrogel microspheres, and control the concentration of calcium ions in the preservation solution to be 3~5mM and the pH value to be 6.5~7.0; when performing in situ injection, adjust the concentration of calcium ions in the preservation solution to 0.5 mM or 2~5 mM and adjust the pH value to 7.0~7.5.

[0009] Furthermore, the raw material components of the lipid nanoparticles described in step (1) are: ionizable lipids, PEG lipids, auxiliary phospholipids, and cholesterol. Ionizable lipids include, but are not limited to, Dlin-MC3-DMA, SM-102, ALC-0315, etc. PEG lipids include, but are not limited to, DSPE-PEG2000, ALC-0159, DMG-PEG2000, etc. Auxiliary lipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine (DSPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-distearyl-sn-glycerol-3-phosphatidylcholine (DSPC), etc.

[0010] Furthermore, in step (1), the lipid nanoparticles are controlled to consist of ionizable lipids, PEG lipids, cofactor phospholipids and cholesterol in a molar ratio of 30~60%: 0~3%: 5~15%: 25~40%, as well as nucleic acids.

[0011] Furthermore, in step (1), the N:P ratio between the lipid nanoparticles and nucleic acids is controlled to be 2~8.

[0012] Furthermore, the concentration of the sodium alginate solution in step (1) is 1~2% w / v.

[0013] Furthermore, the conditions for the gas microfluidic control in step (1) are: nitrogen flow rate of 0.5~2.0 L·min. -1 The sodium alginate flow rate is 15~20 mL / h, the coaxial needle is perpendicular to the ground, and the receiving distance is greater than 5 cm.

[0014] Furthermore, the concentration of the calcium chloride solution in step (1) is 50~500 mM.

[0015] Furthermore, the hydrogel microspheres in step (2) are stored for no more than 6 months.

[0016] The beneficial effects of this invention are as follows: This invention selects sodium alginate to encapsulate LNPs, through ionic crosslinking (Ca... 2+This invention forms hydrogel microspheres (50-200 μm in diameter) that encapsulate LNPs within a three-dimensional network, reducing mechanical stress damage. Ionic cross-linking occurs at room temperature (unlike chitosan, which requires acidic dissolution), making it suitable for GMP continuous production. It enhances local retention and reduces LNP diffusion to non-target tissues. The natural polysaccharide structure can be enzymatically hydrolyzed in vivo, posing no risk of long-term residue. Furthermore, to better ensure LNP stability during preservation, the calcium ion concentration and pH value in the preservation solution are adjusted, significantly extending the preservation time and ensuring LNP stability during storage and transportation. Additionally, by further adjusting the calcium ion concentration and pH value of the preservation solution before local injection of LNP, this invention effectively ensures the activity of LNPs after local injection, achieving highly efficient nucleic acid delivery. Attached Figure Description

[0017] Figure 1 The encapsulation efficiency of LNP by different hydrogel microspheres.

[0018] Figure 2 The cell transfection efficiency after co-culturing different hydrogel microspheres-LNP with cells was determined.

[0019] Figure 3 The effect of different calcium ion concentrations in solution A on the stability of LNPs stored in microspheres.

[0020] Figure 4 The effect of different calcium ion concentrations in solution B on the stability of LNPs stored in microspheres. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention. Example

[0022] I. Experimental Methods 1. Preparation of lipid nanoparticles (LNP) The LNP formulation used in this embodiment mainly includes Dlin-MC3-DMA (AVT Co., CAS: 1224606-06-7), DMG-PEG2000 (AVT Co., CAS: 160743-62-4), DSPC (AVT Co., CAS: 816-94-4), and cholesterol (Aladdin Co., CAS: 57-88-5), but is not limited to this one; any other LNP can be used in the preservation method of this invention.

[0023] The raw materials were prepared according to the molar ratio of Dlin-MC3-DMA, DMG-PEG2000, DSPC, and cholesterol of 50%: 1.5%: 10%: 38.5%. Dlin-MC3-DMA is a dissociable lipid that can be protonated under acidic conditions to form cationic lipids. These cationic lipids bind to negatively charged mRNA via electrostatic interactions, forming mRNA-loaded lipid nanoparticles (LNPs). In the experiment, the above four components were first dissolved in anhydrous ethanol according to the molar ratio, and then the corresponding mass of mRNA was added to a citrate buffer solution based on N / P = 6.

[0024] Further, a microfluidic mixing method (Unigen Biotech Co., Nano S) was used to allow the lipid solution and mRNA solution to fully and rapidly form LNPs with uniform particle size in a micromixer. Since the lipids are dissolved in ethanol and the nucleic acids are dissolved in acidic buffer, residual ethanol was removed by ultrafiltration (30 kD) and the solution system was replaced with PBS.

[0025] 2. Preparation of LNP-hydrogel microspheres First, add the ultrafiltered LNP to the sodium alginate solution and mix thoroughly. Prepare hydrogel microspheres using gas microfluidics. Many factors affect the quality of the hydrogel microspheres, such as nitrogen flow rate, sodium alginate flow rate, sodium alginate concentration, coaxial needle receiving angle, and receiving distance (between the coaxial needle tip and the calcium chloride solution). During preparation, ensure the sodium alginate solution concentration is 1% w / v, as higher concentrations can easily cause needle clogging. Use 1.0 L·min -1 The nitrogen flow rate was maintained at 15-20 mL / h, ensuring the microspheres had good morphology and uniform particle size. The sodium alginate flow rate was kept at 15-20 mL / h. The coaxial needle was perpendicular to the ground, with a receiving distance of at least 5 cm. In short, the sodium alginate solution was injected using a coaxial needle, then sheared into uniform droplets under the influence of the nitrogen flow. The droplets were then dropped into a 100 mM calcium chloride (CaCl2) solution by gravity. The sodium alginate was reacted with CaCl2... 2+ Cross-linking leads to the formation of the core of alginate (calcium alginate) hydrogel microspheres. The alginate hydrogel microspheres were collected, and the remaining ions on the surface were washed twice with ddH2O and stored at 4°C in the dark.

[0026] 3. LNP stability testing (1) DLS detection of LNP: DLS size, Zeta potential, PDI and transmission electron microscopy images of LNP not encapsulated in alginate hydrogel were detected. (2) One month after encapsulation in alginate hydrogel microspheres, the DLS size, Zeta potential, PDI and transmission electron microscopy images of LNPs; (3) Six months after encapsulation in alginate hydrogel microspheres, the DLS size, Zeta potential, PDI and transmission electron microscopy images of LNP.

[0027] II. Experimental Results 1. Stability Study of LNP-Hydrogel Microspheres This invention utilizes gas shear microfluidics to prepare hydrogel microspheres. The inventors previously experimented with encapsulating LNPs with different hydrogels to achieve both LNP stability and rapid release. We selected different hydrogels, including alginate, hyaluronic acid, chitosan, and gelatin, for encapsulation experiments. The specific results are shown in Table 1.

[0028] Table 1

[0029] As shown in Table 1, this invention selects an alginate hydrogel microsphere encapsulation method to preserve LNPs. Alginate is mainly cross-linked by calcium ions, allowing droplets to directly fall into the Ca2+ ion matrix. 2+ Rapid sphere formation in solution (second-level solidification) is suitable for continuous production in gas shear microfluidics. The microspheres swell easily in physiological environments, making them superior in all aspects.

[0030] When using hyaluronic acid hydrogels, it is necessary to use a composite ionic crosslinking agent (such as Ca). 2+ Methods involving chemical cross-linking (such as EDC / NHS) present complex processing issues. While using chitosan to adsorb negatively charged LNPs or drugs can reduce burst release, it requires an acidic solvent (such as 1% acetic acid) to release the LNPs, which affects their stability. Furthermore, when using gelatin microspheres to preserve LNPs, the hydrogel is prone to disintegration at 37°C, necessitating chemical cross-linking (such as GelMA photocross-linking or genipin) to enhance stability.

[0031] 2. Study on the encapsulation efficiency and transfection efficiency of LNP by hydrogel microspheres The inventors compared the encapsulation efficiency of LNP with different hydrogel microspheres, and the results are as follows: Figure 1 As shown, calcium alginate microspheres were found to have the highest encapsulation efficiency.

[0032] Further co-culturing of four different microspheres with cells yielded the following results: Figure 2 As shown, the cell transfection efficiency was highest when cells were co-cultured with sodium alginate microspheres.

[0033] 3. Study on the preservation stability of LNP-hydrogel microspheres To enhance the stability of alginate microspheres for LNP, solution A was used for preservation during the storage of alginate microsphere-LNP. Solution A contained a calcium ion concentration of 3-5 mM and maintained a pH between 6.5 and 7.0. However, when performing in situ injection, the inventors adjusted solution A to solution B, reducing the calcium ion concentration to 1-5 mM and maintaining a pH between 7.2 and 7.4.

[0034] The effect of different calcium ion concentrations in solution A on the stability of LNP was tested, and the results are as follows: Figure 3 As shown, when the calcium ion concentration is 3-5 mM, the transfection efficiency of LNP after one week is significantly higher than that when the calcium ion concentration is 1 and 2 mM. Moreover, when the calcium ion concentration is 3 mM, the transfection efficiency of LNP is the best after one week at 4℃.

[0035] 4. Stability study of LNP-hydrogel microspheres before injection use The disintegration of alginate microspheres in solution B under different calcium ion concentrations was tested, and the effect of 3mM Ca on the disintegration of the microspheres was investigated. Cross-linked microspheres formed in 0.5 mM to 5 mM Ca The disintegration behavior in the solution (solution B) was dynamically monitored. The results were obtained by recording the remaining integrity (%) of the microspheres at different time points, as shown below. Figure 4 As shown. Figure 4 The results showed that the microspheres were in 1mM Ca Under these conditions, the disintegration rate was significantly faster than other groups; the integrity dropped sharply to 60% in just 5 minutes, further decreased to 10% at 10 minutes, and completely disintegrated (0%) after 20 minutes, indicating that Ca2+ disintegration under this concentration gradient was significantly faster than in other groups. 2+ The migration inside and outside the microspheres was the most intense, rapidly destroying the original cross-linked structure. In contrast, the microsphere structures remained relatively stable in the other concentration groups (0.5, 2, 3, 4, and 5 mM), with the integrity remaining above 70% within 60 minutes.

Claims

1. A method for preserving and using locally injected lipid nanoparticles, characterized in that, Includes the following steps: (1) Mix LNP with sodium alginate solution evenly, and then prepare hydrogel microspheres by gas shear microfluidic method, and collect the hydrogel microspheres in calcium ion solution; (2) Use a storage solution containing calcium ions to store hydrogel microspheres, and control the concentration of calcium ions in the storage solution to be 3~5 mM and the pH value to be 6.5~7.0; when performing in situ injection, adjust the concentration of calcium ions in the storage solution to 0.5 mM or 2~5 mM and adjust the pH value to 7.0~7.

5.

2. The method according to claim 1, characterized in that, The raw material components of LNP in step (1) are: ionizable lipids, PEG lipids, cofactor phospholipids and cholesterol.

3. The method according to claim 2, characterized in that, Ionizable lipids include Dlin-MC3-DMA, SM-102, or ALC-0315.

4. The method according to claim 2, characterized in that, PEG lipids include DSPE-PEG2000, ALC-0159, and DMG-PEG2000; auxiliary lipids include 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, or 1,2-distearyl-sn-glycerol-3-phosphatidylcholine.

5. The method according to claim 2, characterized in that, In step (1), ionizable lipids, PEG lipids, cofactor phospholipids and cholesterol are composed in a molar ratio of 30~60%: 0~3%: 5~15%: 25~40%, as well as nucleic acids.

6. The method according to claim 2, characterized in that, In step (1), the N:P ratio between LNP and nucleic acid is controlled to be 2~8.

7. The method according to claim 1, characterized in that, The concentration of the sodium alginate solution mentioned in step (1) is 1~2% w / v.

8. The method according to claim 1, characterized in that, The conditions for gas microfluidics described in step (1) are: nitrogen flow rate of 0.5~2.0 L·min. -1 The sodium alginate flow rate is 15~20 mL / h, the coaxial needle is perpendicular to the ground, and the receiving distance is greater than 5cm.

9. The method according to claim 1, characterized in that, The Ca mentioned in step (1) 2+ The concentration of the solution is 50~500mM.

10. The method according to claim 1, characterized in that, In step (2), the hydrogel microspheres should be stored for no more than 6 months.

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