Lipid nanoparticle freeze-drying protective agent composition containing mucus dissolving agent, lipid nanoparticle preparation and application

Abstract

The invention discloses a lipid nanoparticle freeze-drying protective agent composition containing a mucus dissolving agent, a lipid nanoparticle preparation and application, and belongs to the technical field of biological medicine, and the freeze-drying protective agent composition comprises saccharides and the mucus dissolving agent. According to the invention, the saccharide and the mucus dissolving agent are combined to form a freeze-drying protective agent composition system for the first time. Through the synergistic effect of the carbohydrates and the mucus dissolving agent, the lipid nanoparticles can be stably stored at room temperature after being freeze-dried, a cold chain is not needed, the storage and transportation cost is remarkably reduced, and the clinical application convenience of the preparation is improved. In an in-vitro mucus model and an in-vivo mouse asthma model, it is proved that the medicine delivery efficiency of the lung can be effectively improved, and the treatment effect is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a lyophilization protectant composition for stabilizing lipid nanoparticles. Specifically, it relates to a composite lyophilization protectant comprising sugars and mucolytics, which is used for the lyophilization of lipid nanoparticles encapsulating therapeutic loads to improve their stability, promote their pulmonary mucosal permeability, and for the treatment of lung diseases. Background Technology

[0002] Lipid nanoparticles (LNPs), as key delivery carriers for nucleic acid drugs (such as mRNA and siRNA), have advantages such as good biocompatibility, high delivery efficiency, and protection of nucleic acids from degradation, and are widely used in the biomedical field. However, lung delivery of LNPs still faces many key challenges, among which two key technological bottlenecks are particularly significant: On the one hand, LNPs loaded with therapeutic nucleic acids have poor stability in the liquid state, the particles are prone to aggregation and the lipid components are prone to oxidation. They usually require low-temperature cold chain (such as -20°C or -80°C) storage and transportation, which greatly increases the storage cost, transportation difficulty and clinical application threshold of the formulation, and limits its accessibility in resource-limited areas.

[0003] On the other hand, many nucleic acid drugs need to act on mucosal tissues such as the lungs. However, the mucus layer on the surface of the mucosa (especially with increased mucus secretion in diseased states) forms a physical barrier, severely hindering the penetration of lipid nanoparticles and resulting in ineffective drug delivery. Taking lung diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis as examples, patients have abnormally increased and viscous mucus secretion in their lungs, making it difficult for traditional LNP formulations to penetrate the mucus layer and reach target cells, ultimately leading to a significant decrease in drug delivery efficiency.

[0004] Freeze-drying technology is an effective strategy for addressing the stability issues of liquid nanoparticles (LNPs). This technology can transform liquid formulations into a solid form, thereby achieving long-term stable storage of the formulation at 2℃-8℃. Currently, the freeze-drying protectants used in LNPs are mostly sugars (such as sucrose and trehalose). These protectants maintain the stability of the nanostructure by replacing water molecules and forming hydrogen bonds with the lipid membrane during the drying process. However, most existing freeze-drying systems only focus on improving storage stability and fail to address the mucosal penetration problem during drug delivery.

[0005] Existing technologies (such as Lababidi et al., 2020, https: / / doi.org / 10.1016 / j.ejpb.2020.10.010) disclose the use of the mucolytic N-acetylcysteine ​​(NAC) blended with antibiotics to prepare spray-dried PLGA nanoparticle inhalation formulations, thereby enhancing the mucus penetration capacity of the formulations. However, this technology still has limitations: firstly, it does not provide solutions to specific problems in the lyophilization process of LNPs; secondly, compared with PLGA nanoparticles, the core-shell structure of LNPs is more sensitive to various stresses (such as ice crystal damage and osmotic stress) during the lyophilization process, and simple component combinations cannot solve the dual challenges it faces in terms of stability and functionality.

[0006] Therefore, there is an urgent need in this field to develop a novel lyophilization protectant composition that not only achieves storage stability of lipid nanoparticles at room temperature but also endows lyophilized LNPs with the ability to overcome the mucus barrier. This technology is of great significance for promoting the application of nucleic acid drugs in the inhalation therapy of lung-related diseases. Summary of the Invention

[0007] The primary objective of this invention is to provide a lipid nanoparticle lyophilization protectant composition containing a mucolytic agent, thereby solving the dual technical challenges of storage stability and lung mucosal permeability in existing lipid nanoparticle formulations.

[0008] A second objective of the present invention is to provide a method for preparing lyophilized LNPs formulations using the lyophilization protectant composition.

[0009] A third objective of this invention is to provide the use of lyophilized LNPs formulations prepared using the lyophilization protectant composition in the preparation of medicaments for treating lung diseases.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A lipid nanoparticle lyophilization protectant composition containing a mucolytic agent is characterized by comprising two key components: a carbohydrate component and a mucolytic agent component. Wherein: Sugar component: Selected from one or more sugars, serving as the base component of the lyophilization protectant to maintain the structural integrity of lipid nanoparticles during lyophilization and reconstitution. The sugar is selected from at least one of sucrose, trehalose, mannitol, lactose, and glucose. Sucrose and trehalose are preferred, both possessing excellent glass-forming ability and dehydration protection. The sugar, as one component of the lyophilization protectant composition, is used to prepare lyophilized LNPs formulations; its final concentration in the LNPs formulation system is 5%-20% (…). w / v ).

[0011] Mucolytic agent component: Used to disrupt the mucus barrier of the mucosa and enhance the mucus penetration ability of lipid nanoparticles. The mucolytic agent is selected from at least one of N-acetylcysteine ​​(NAC), erdocysteine, stironine, bromhexine, ambroxol, carbocysteine, and recombinant human deoxyribonuclease. N-acetylcysteine ​​(NAC) is preferred, as its safety has been clinically verified and it has definite mucolytic activity. The mucolytic agent, as one component of the lyophilization protectant composition, is used to prepare lyophilized LNPs formulations; its final concentration in the LNPs formulation system is 0.5%-5% (…). w / v ).

[0012] A lipid nanoparticle or lipid nanoparticle group encapsulated with a therapeutic load is formulated and freeze-dried using the lipid nanoparticle lyophilization protectant composition containing a mucolytic agent described in this invention.

[0013] The therapeutic payload is a nucleic acid, which is mRNA, siRNA, miRNA, plasmid DNA, antisense oligonucleotide, or a component of the CRISPR / Cas system.

[0014] The preparation method of the lipid nanoparticles or lipid nanoparticle groups is achieved through the following steps: preparing nucleic acid-loaded lipid nanoparticles using microfluidics; using tris(hydroxymethyl)aminomethane (Tris) buffer as a solvent to dissolve sugars and mucolytic agents, and preparing a lyophilization protectant composition solution; mixing the nucleic acid-loaded lipid nanoparticles with the prepared lyophilization protectant composition solution evenly, and performing freeze-drying treatment to obtain the lyophilized LNPs formulation.

[0015] Furthermore, in the mixture of nucleic acid-loaded lipid nanoparticles and the prepared lyophilization protectant composition solution, the final concentration of sugars (such as sucrose) is 5%-20%. w / v The final concentration of the mucolytic agent (such as NAC) is 0.5%-5%. w / v ).

[0016] Furthermore, the freeze-drying process can employ conventional freeze-drying techniques in the art. The lipid nanoparticles can be freeze-dried to form a freeze-dried cake. The freeze-dried formulation can be stored stably at room temperature and can be reconstituted before application by adding a reconstitution medium. The reconstitution medium is nuclease-free pure water or nuclease-free phosphate buffer (PBS, typically pH 7.2-7.4, 0.01 M, depending on the formulation requirements).

[0017] Further, the preparation method of the lipid nanoparticles or lipid nanoparticle groups is as follows: Lipid nanoparticles loaded with nucleic acids are prepared using microfluidic technology. Sugars and a mucolytic agent are weighed and dissolved in a solvent, wherein the solvent is tris(hydroxymethyl)aminomethane (Tris) buffer. The solution is then filtered through a 0.22 μm sterile filter to obtain a lipid nanoparticle lyophilization protectant composition solution containing the mucolytic agent. This lyophilization protectant composition solution is mixed evenly with the nucleic acid-loaded lipid nanoparticles and dispensed into vials. After dispensing, a freeze-drying process is performed. The freeze-drying process is as follows: Pre-freezing stage temperature is -35℃, pre-freezing time is 5 hours; primary drying stage temperature is -20℃, time is 10 hours, vacuum pressure is controlled at ≤10 Pa; secondary drying stage temperature is 2℃, time is 5 hours, vacuum pressure is controlled at ≤10 Pa; final temperature is 25℃, maintained for 5 hours, for a total duration of approximately 25 hours, yielding the freeze-dried LNPs formulation of this invention.

[0018] A pharmaceutical composition comprising the lyophilized lipid nanoparticles described herein and a pharmaceutically acceptable carrier. The dosage form of the pharmaceutical composition is an inhalation formulation, such as a nebulized inhalation liquid or dry powder inhaler, or may be an injection or oral formulation.

[0019] The lyophilized lipid nanoparticle formulation or the pharmaceutical composition described in this invention is used to prepare a medicament for the prevention or treatment of lung diseases. These lung diseases include asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), lung infections, and lung cancer.

[0020] Compared with the prior art, the present invention has the following significant advantages: 1. Overcoming the limitations of low-temperature storage: This invention is the first to combine sugars (acting as freeze-drying protectants) with mucolytic agents (acting as penetration enhancers) to form a freeze-drying protectant composition system. Through the synergistic effect of sugars and mucolytic agents, lipid nanoparticles can be stably stored at room temperature after freeze-drying, without the need for a cold chain, significantly reducing storage and transportation costs and improving the convenience of clinical application of the formulation.

[0021] 2. Enhances mucus penetration and therapeutic efficacy: The mucolytic agent in the lyophilized formulation can act directly on the mucus layer after the formulation is reconstituted, reducing mucus viscosity and promoting the penetration of lipid nanoparticles through the mucus barrier to reach the target tissue. For example, it has been shown to effectively improve the efficiency of drug delivery to the lungs and enhance the therapeutic effect in both in vitro mucus models and in vivo mouse asthma models.

[0022] 3. Excellent formulation stability: The combination of specific concentrations of sugars and mucolytic agents can maintain the structural stability of lipid nanoparticles during freeze-drying, storage and reconstitution, ensuring the integrity and activity of nucleic acid carriers. After reconstitution, the formulation has uniform particle size and high encapsulation efficiency.

[0023] 4. Good safety and applicability: The sugars (such as sucrose) and mucolytics (such as N-acetylcysteine) used are all commonly used excipients or drug components in clinical practice, with good biocompatibility; the formulation can be precisely delivered to the lungs through inhalation, and is especially suitable for the treatment of mucus-related lung diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Attached Figure Description

[0024] Figure 1 These are photographs of the appearance of lipid nanoparticles prepared in Example 1 and Comparative Examples 1-3 of this invention after freeze-drying.

[0025] Figure 2 This is a comparison of the particle size (A), polydispersity index (PDI) (B), and encapsulation efficiency (C) of the lipid nanoparticles prepared in Example 1 and Comparative Examples 1-3 of this invention before freeze-drying and after freeze-drying and rehydration.

[0026] Figure 3 In this invention, the final sucrose concentration is fixed at 10%. w / v The effect of different final NAC concentrations on the particle size of lyophilized LNPs formulations after rehydration.

[0027] Figure 4 In this invention, the final NAC concentration is fixed at 1%. w / v The effect of different final sucrose concentrations on the particle size of rehydrated freeze-dried LNPs formulations.

[0028] Figure 5 This is a representative trajectory diagram of the movement of the lyophilized and reconstituted LNPs formulations prepared in Example 1 and Comparative Example 3 of this invention in in vitro artificial mucus, as determined by multi-particle tracking technology.

[0029] Figure 6 Based on the present invention Figure 5 The statistical analysis results of the mean square displacement (MSD) of the two types of LNPs in the artificial mucus in vitro were obtained from multi-particle tracking data.

[0030] Figure 7 This study evaluates the therapeutic effect of the lyophilized LNPs formulation in an ovalbumin (OVA)-induced mouse asthma model. A represents the lung slice inflammation score, B represents the collagen volume fraction, and C represents the relative number of goblet cells.

[0031] Figure 8 This is an evaluation of the long-term stability of the lyophilized LNPs formulation in this invention under different storage temperatures; where A: average particle size change, B: polydispersity index change, and C: encapsulation efficiency change. Detailed Implementation

[0032] TSLP-siRNA: TSLP, or Thymic Stromal Lymphopoietin, is a class of cytokines primarily secreted by epithelial cells. Its core function is to initiate and regulate allergic inflammatory responses, especially in the pathogenesis of allergic airway inflammation (such as asthma and chronic obstructive pulmonary disease), where it can activate immune cells to mediate inflammatory cascades. The TSLP-siRNA used in this invention is Mouse TSLP siRNA (technology type: ON-TARGETplus, SMARTpool), purchased from Dharmacon, USA. This reagent consists of four sequences, the sequence information of which is as follows: Target sequence 1: AAUGAGCAAUAGACCGUUA (SEQ ID NO.1); Target sequence 2: GAAAAUGACAGUUCGGGCA (SEQ ID NO.2); Target sequence 3: UGAGAGAAAUGACGGUACU (SEQ ID NO.3); Target sequence 4: CAGUAUAGGUGCUUUAAAU (SEQ ID NO. 4).

[0033] Example 1

[0034] Preparation of lyophilization protectant compositions and lyophilized LNP formulations: 2 g of sucrose and 0.5 g of N-acetylcysteine ​​(NAC) were weighed and dissolved in 10 mL of a Tris buffer solution. The solution was then filtered through a 0.22 μm sterile filter to obtain a lyophilization protectant composition solution. Lipid nanoparticles encapsulating therapeutic small interfering RNA (siRNA) were prepared using microfluidic technology. The siRNA encapsulates a target gene (TSLP-siRNA) related to lung disease treatment and was used as a model sample. The specific preparation method for lipid nanoparticles (LNPs) involved combining ionizable cationic lipids (Dlin-MC3-DMA), phospholipids (DSPC), cholesterol, and PEGylated lipids (DMG-PEG). 2000The siRNA stock solution was dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to obtain an ethanol solution of the lipid phase. The siRNA stock solution was diluted to the desired concentration with an acidic buffer (acetic acid-sodium acetate buffer, pH=4.0, 10 mM). At room temperature, using a commercial microfluidic instrument (e.g., the microfluidic preparation instrument from NavInfo (Shanghai) Nanotechnology Co., Ltd.), the lipid phase and aqueous phase were pumped into a microfluidic chip at flow rates of 3 mL / min and 9 mL / min, respectively, for mixing. The volume ratio of the two phases (organic phase: aqueous phase) was controlled at 1:3 to obtain a fresh LNP dispersion. The fresh LNP dispersion was buffer exchanged and concentrated using ultrafiltration centrifuge tubes with a molecular weight cutoff (MWCO) of 50 kDa, using Tris buffer as the replacement buffer. This aimed to remove residual ethanol and stabilize the LNPs in an aqueous environment suitable for freeze-drying, obtaining a siRNA-LNP stock solution suitable for subsequent freeze-drying. Take 1.0 mL of the model sample siRNA-LNPs stock solution and mix it with an equal volume of 1.0 mL of the above-described lyophilization protectant composition solution, mixing gently until homogeneous. At this point, the final concentration of sucrose in the mixture is 100 mg / mL (i.e., 10%). w / v The final concentration of NAC was 25 mg / mL (i.e., 2.5%). w / v The mixture was dispensed into vials for subsequent freeze-drying. After dispensing, a freeze-drying procedure was employed (pre-cooling → primary drying → secondary drying; pre-freezing temperature -35°C, pre-freezing time 5 hours; primary drying temperature -20°C, time 10 hours, vacuum pressure controlled ≤10 Pa; secondary drying temperature 2°C, time 5 hours, vacuum pressure controlled ≤10 Pa; final temperature 25°C, maintained for 5 hours) for a total freeze-drying time of approximately 25 hours, yielding the freeze-dried LNPs formulation described in this invention. Figure 1 ).

[0035] Comparative Example 1: No freeze-drying protectant was added, and the LNPs were prepared and freeze-dried in the same manner as in Example 1.

[0036] Comparative Example 2: Only NAC was used as the freeze-drying protectant, and the preparation and freeze-drying procedures of LNPs were the same as in Example 1.

[0037] Comparative Example 3: Sucrose was used as the freeze-drying protectant only. The preparation and freeze-drying procedures for LNPs were the same as in Example 1.

[0038] Example 2

[0039] Key physicochemical characterization properties of lyophilized LNP formulations: The freeze-dried cakes obtained in Example 1 and Comparative Examples 1-3 were reconstituted with nuclease-free pure water, gently shaken, and allowed to stand for 10 minutes. Particle size and polydispersity index (PDI) were measured at 25°C using a dynamic light scattering (DLS) particle size analyzer (e.g., Malvern Zetasizer Nano ZS90). Each sample was measured three times, and the average value was taken. A comparison chart of particle size and PDI was plotted. Figure 2 A, 2B). The encapsulation efficiency of siRNA was determined using a standard method based on RiboGreen fluorescence staining, and the results were plotted as a bar chart. Figure 2 C). The original LNPs liquid formulation that has not undergone lyophilization is used as a reference.

[0040] The results are as follows Figure 2 As shown: After reconstitution, the average particle size, PDI, and encapsulation efficiency of the lyophilized LNPs formulation in Example 1 of this invention showed no significant changes compared to the original solution before lyophilization, and no significant differences compared to Comparative Example 3 (sucrose protectant only). This indicates that the lyophilization protectant composition of this invention can excellently maintain the nanostructure integrity of LNPs and the encapsulation state of siRNA.

[0041] In contrast, the freeze-dried cakes of Comparative Example 1 (without freeze-drying protectant) and Comparative Example 2 (with NAC protectant only) showed significant aggregation after reconstitution, with a significant increase in average particle size and PDI, and a significant decrease in encapsulation efficiency, proving that NAC is not suitable as a single freeze-drying protectant for LNPs.

[0042] Example 3 Following the method of Example 1, in the mixture of LNPs and the lyophilization protectant composition, the final sucrose concentration was fixed at 10%. w / v Combined with different final concentrations of NAC (0.5%, 1%, 1.5%, 2%, 5%) w / v The lyophilized LNPs formulation was prepared, and the particle size test results of the lyophilized formulation are as follows: Figure 3 As shown.

[0043] Following the method of Example 1, in the mixture system of LNPs and lyophilization protectant composition, the final NAC concentration was fixed at 1%. w / v This is combined with different final concentrations of sucrose (5%, 10%, 15%, 20%). w / v The lyophilized LNPs formulation was prepared, and the particle size test results of the lyophilized formulation are as follows: Figure 4 As shown.

[0044] Example 4 In vitro mucus penetration assessment of lyophilized LNP formulations: The ability of lyophilized LNPs to penetrate the mucus barrier was evaluated using multi-particle tracking (MPT) analysis. Artificial mucus was prepared and placed in a confocal culture dish. Lyophilized LNP formulations from Example 1 (lyophilization protectant composition) and Comparative Example 3 (sucrose protectant only) were reconstituted and added to the surface of the artificial mucus. Real-time tracking was performed using MPT under a laser confocal microscope, recording the particle motion videos. The particle trajectories and relative displacements (RDs) were analyzed using specialized software. Figure 5 ), calculate its mean square displacement (MSD) ( Figure 6 The original LNPs liquid formulation that has not undergone lyophilization was used as a reference.

[0045] The results are as follows Figure 5-6 As shown, the lyophilized LNPs formulation of Example 1 of this invention exhibits a significantly higher MSD value, indicating stronger motility and less restriction in mucus. This demonstrates that the NAC bound to the LNPs during lyophilization can more effectively dissolve mucus upon contact, opening permeation channels for the LNPs. In contrast, the LNPs in Comparative Example 3 showed significantly restricted motility, proving that traditional sucrose lyophilization protectants cannot achieve the mucus permeation effect described in this invention.

[0046] Example 5 Therapeutic effects of lyophilized LNPs formulations in a mouse asthma model: A BALB / c mouse asthma model was established using ovalbumin (OVA) sensitization and challenge. Mice weighed 18–22 g. Mice were randomly divided into 5 groups of 6 mice each: (1) healthy group; (2) asthma model group (sensitization and challenge only, no drug administration); (3) liquid LNPs preparation group (inhalation administration of the original, un-lyophilized liquid LNPs); (4) Comparative Example 3 group (inhalation administration of lyophilized reconstituted LNPs containing only sucrose protectant); (5) Example 1 group (inhalation administration of lyophilized reconstituted LNPs containing the lyophilized protectant composition of this invention). All LNPs in each group were encapsulated with an equal dose of therapeutic siRNA. OVA sensitization phase: OVA and Al(OH)3 suspension were injected intraperitoneally on days 0, 7, and 14; OVA challenge phase: OVA solution was nebulized for 30 minutes daily from days 21 to 28; In the treatment group, the corresponding formulation was administered via a handheld mouse lung nebulizer on days 24, 26, and 28, at a dose of 4 μg siRNA per mouse. Twenty-four hours after the last administration, mouse lung tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and semi-quantitative pathological scoring was performed. Figure 7 A); Masson staining was used to evaluate the proliferation of collagen fibers in the airway wall (A); Figure 7B), goblet cell proliferation was evaluated using AB-PAS staining. Figure 7 C), draw the corresponding bar chart.

[0047] The results are as follows Figure 7 As shown, the asthma model group exhibited significant asthma pathological features: inflammatory cell infiltration, increased mucus secretion, and goblet cell proliferation. Comparative Group 3 (inhaled administration of lyophilized reconstituted LNPs containing only sucrose protectant) showed some therapeutic effect, but the improvement in mucus secretion was limited. In contrast, Group 1 (lyophilized reconstituted LNPs containing the lyophilized protectant composition of this invention) showed the most significant therapeutic effect, with reduced inflammatory pathological scores, decreased collagen deposition, and weakened goblet cell proliferation. This fully demonstrates that the lyophilized LNPs preparation prepared by the method of this invention, possessing both stability and mucus permeability, can effectively deliver therapeutic nucleic acids to the lesion, thereby significantly enhancing the therapeutic effect on lung diseases.

[0048] Example 6 Long-term stability study of lyophilized LNP formulations: To verify whether the lyophilized LNPs formulation prepared using the lyophilization protectant composition described in this invention can be stored stably for a long period at different temperatures, especially at room temperature, the following long-term stability experiment was conducted. The lyophilized LNPs formulation prepared in Example 1 (using the lyophilization protectant composition described in this invention) was stored at -20°C, 4°C, and 25°C, respectively. Samples were taken at each preset time point (0, 1, 3, and 6 months) for testing, and key physicochemical properties were characterized according to the method in Example 2.

[0049] The results are as follows Figure 8 As shown, the lyophilized LNPs formulation prepared in Example 1 of this invention exhibits long-term stability. After storage at room temperature (25°C) for 6 months, its particle size only slightly increased (from 109.1 nm to 129.4 nm), the PDI remained below 0.2, and the encapsulation efficiency of siRNA remained above 80% (82.5%). This indicates that the lyophilization protectant composition of this invention can effectively protect the LNPs structure and siRNA load, enabling it to be stored stably at room temperature for a long period.

Claims

1. A mucus-dissolving agent-containing lipid nanoparticle lyophilization protectant composition, characterized by, The sugar is at least one selected from sucrose, trehalose, lactose, glucose, mannitol; the mucolytic agent is at least one selected from N-acetylcysteine, erdosteine, domidium, bromhexine, ambroxol, carbocysteine, recombinant human deoxyribonuclease.

2. The mucus dissolver-containing lipid nanoparticle lyophilization cryoprotectant composition of claim 1, wherein, w / v 3. The mucus dissolver-containing lipid nanoparticle lyophilization cryoprotectant composition of claim 1, wherein, The sugar has a final concentration of 5-20% in the formulation system when used for preparing the freeze-dried lipid nanoparticle formulation The mucolytic agent-containing lipid nanoparticle lyophilization protectant composition of any one of claims 1-3 is used for preparation. The mucolytic agent has a final concentration of 0.5-5% in the formulation system when used for preparing the freeze-dried lipid nanoparticle formulation w / v ).

4. A lipid nanoparticle or population of lipid nanoparticles encapsulating a therapeutic payload nucleic acid, characterized in that, The nucleic acid is mRNA, siRNA, miRNA, plasmid DNA, antisense oligonucleotide, or CRISPR / Cas system component.

5. The lipid nanoparticle or population of lipid nanoparticles of claim 4, wherein, The method comprises the following steps: preparing lipid nanoparticles by using a microfluidic method; 6. A method of preparing the lipid nanoparticle or population of lipid nanoparticles of any one of claims 4-5, characterized in that, The prepared lipid nanoparticles are mixed with the prepared lyophilization protectant composition solution, and then subjected to freeze-drying treatment to obtain a lyophilized lipid nanoparticle preparation. w / v 7. The method of claim 6, wherein the lipid nanoparticle or population of lipid nanoparticles is prepared by a method comprising: In the lipid nanoparticle formulation system, the final concentration of the sugar is 5%-20% w / v , the final concentration of the mucolytic agent is 0.5%-5% The pharmaceutical composition comprises the lipid nanoparticle or population of lipid nanoparticles of any one of claims 4-5 and a pharmaceutically acceptable carrier; the dosage form of the pharmaceutical composition is an inhalant, dry powder inhalant, or atomized liquid. ; the freeze-drying procedure is: the pre-freezing stage temperature is -35℃, the pre-freezing time is 5 hours; the first drying stage temperature is -20℃, the time is 10 hours, and the vacuum pressure is controlled to be ≤10 Pa; the second drying stage temperature is 2℃, the time is 5 hours, and the vacuum pressure is controlled to be ≤10 Pa; the end point temperature is 25℃, and the maintenance time is 5 hours.

8. A pharmaceutical composition, characterized by, 9. Use of the lipid nanoparticle or population of lipid nanoparticles of any one of claims 4-5 or the pharmaceutical composition of claim 8 in the preparation of a medicament for preventing and / or treating a pulmonary disease. The pulmonary disease is selected from asthma, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary infection, lung cancer.

10. Use according to claim 9, characterized in that, ​

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