Permanent cationic lipids to improve stability of aerosolisable lipid nanoparticles and uses thereof

Abstract

The application provides a kind of permanent cationic lipid for improving the stability of aerosolizable lipid nanoparticles and its application, which is composed of two long fatty chains, a quaternary amine head group and an intermediate linker. By changing part of the intermediate linker, the distance between the quaternary amine head group and the linker is adjusted, and the target compound is screened. The technical problem that the positive charge lipid in the prior art is difficult to balance the lung targeting and aerosol stability is solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical delivery technology, and in particular to a permanent cationic lipid for improving the stability of atomizable lipid nanoparticles and its applications. Background Technology

[0002] Messenger RNA (mRNA) vaccines, as a new generation of preventive and therapeutic biological agents, have demonstrated enormous application potential and clinical value in infectious disease control and cancer treatment due to their advantages such as short development cycles, high immune responses, and controllable safety. However, mRNA itself suffers from problems such as susceptibility to nuclease degradation, poor cell membrane penetration, and the potential to trigger immune clearance. Successful in vivo delivery highly depends on a safe and efficient vector system. Among these, lipid nanoparticles (LNPs) are currently the most mature and widely used non-viral delivery vector in clinical translation globally.

[0003] Conventional LNP formulations are mainly composed of four components: ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-modified lipids. They can encapsulate and protect mRNA from nuclease degradation through a lipid bilayer structure, while also promoting mRNA endocytosis and endosome escape through pH-responsive properties, ultimately achieving efficient expression of the target protein.

[0004] For respiratory pathogens such as respiratory syncytial virus (RSV), influenza virus, and COVID-19, mRNA vaccines can be delivered directly to the respiratory mucosa and lung tissue via nebulized inhalation. This can stimulate strong local mucosal immunity at the infection portal site, while also inducing systemic humoral and cellular immunity. Compared with traditional injection administration, this method has a more direct protective effect and is an ideal strategy for the development of vaccines for respiratory infectious diseases.

[0005] However, the application of LNP formulations in nebulized inhalation still faces significant technical bottlenecks. On the one hand, lung epithelial cells and mucosal surfaces are inherently negatively charged. Based on the principle of electrostatic interaction, introducing permanently positively charged lipids into LNP formulations can enhance the binding ability of LNPs to lung tissue, improving their enrichment efficiency and cellular uptake levels. Numerous academic studies and patent documents have confirmed that incorporating commercially available permanently cationic lipids such as DOTAP and EPC as cofactors into LNP formulations can significantly improve mRNA expression efficiency in lung tissue, representing a mainstream technological direction for optimizing lung-targeted delivery. On the other hand, nebulization is an extreme physical process involving high energy and high shear forces. This process easily disrupts the integrity of the lipid bilayer structure of LN, leading to problems such as mRNA leakage, particle aggregation, abnormally large particle size, and decreased dispersion uniformity, ultimately severely weakening vaccine delivery efficiency and medication safety. While existing commercially available permanent cationic lipids optimize the lung targeting of LNPs, they significantly alter the surface potential and lipid membrane fluidity of LNPs, disrupting the compatibility between lipid components. This results in a substantial decrease in the physical stability, storage stability, and redispersibility of LNPs before and after nebulization, failing to meet the formulation requirements for nebulized administration and severely restricting the practical application of this technology in inhaled mRNA vaccines.

[0006] In summary, a core technological contradiction in this field urgently needs to be resolved: the incompatibility between the need to improve the lung-targeted delivery efficiency of LNP formulations and the need to maintain structural stability during nebulization. Specifically, existing commercially available permanent cationic lipids cannot achieve an effective balance between lung targeting and nebulization stability; currently, there are no dedicated cationic lipid components suitable for nebulized LNP delivery systems. Summary of the Invention

[0007] The purpose of this invention is to provide a permanent cationic lipid and its application to improve the stability of atomizable lipid nanoparticles, particularly suitable for atomized drug delivery LNP systems, in order to solve the technical problem in the prior art that positively charged lipids are difficult to balance lung targeting and atomization stability.

[0008] To achieve the above objectives, this technical solution provides a permanent cationic lipid that improves the stability of atomizable lipid nanoparticles, the general structural formula of which is shown below: ; Wherein R1 is a C6~C20 alkyl group or an alkenyl group; R2 is a C1~C3 alkyl group; R3 and R4 are hydrogen or C1~C2 alkyl groups; R5 is a C6~C20 alkyl group or contains an alkenyl group; R6 is a C1~C6 alkyl group; R7, R8, and R9 are C1-C3 alkyl groups; X-: Pharmaceutically acceptable anion.

[0009] It should be noted that alkenyl groups refer to unsaturated aliphatic hydrocarbon monovalent groups containing at least one carbon-carbon double bond (C=C) in the carbon chain. That is, C6~C20 alkenyl groups refer to unsaturated aliphatic hydrocarbon monovalent groups with a total number of carbon atoms between 6 and 20, containing at least one carbon-carbon double bond (C=C) in the carbon chain.

[0010] The permanent cationic lipid provided in this solution, which enhances the stability of atomizable lipid nanoparticles, consists of two long fatty acid chains, a quaternary ammonium head group, and an intermediate linker group. By modifying the intermediate linker portion and adjusting the distance from the quaternary ammonium head group to the linker, target compounds can be screened. This solves the technical problem in existing technologies where positively charged lipids cannot simultaneously achieve lung targeting and atomization stability. It not only needs to effectively improve the lung targeting and transfection efficiency of LNPs, but more importantly, it can ensure that the LNPs formed maintain excellent physicochemical stability (including particle size distribution, encapsulation efficiency, and dispersion uniformity) before, during, and after atomization, thereby truly meeting the stringent requirements of inhaled mRNA vaccine formulations.

[0011] Specifically, the permanently cationic lipids provided in this scheme possess quaternary ammonium salt groups, and the positively charged quaternary ammonium head group can form a stable electrostatic binding with negatively charged mRNA. The quaternary ammonium salt group is a pH-insensitive, permanently positively charged center, maintaining stable positive charge under physiological pH, during nebulization, and in the respiratory mucosal environment. Meanwhile, negatively charged sialic acid residues and glycosaminoglycans are widely distributed on the respiratory epithelial cells and mucosal surface of lung tissue. Through electrostatic interaction, these components significantly enhance the adhesion, accumulation, and retention efficiency of LNPs in the lungs, preventing rapid clearance of LNPs by mucus or entry into systemic circulation, fundamentally improving lung targeting. Furthermore, the positively charged quaternary ammonium head group of the permanently cationic lipids provided in this scheme is connected to the molecular backbone via a flexible linker containing an ester group. Simultaneously, the distance between the quaternary ammonium head group and the hydrophobic backbone is controlled to avoid excessive charge concentration.

[0012] Our team discovered that in traditional DOTAPs, there is only a very short linker between the positively charged quaternary ammonium head group and the two hydrophobic fatty tail chains. The positively charged head group is almost directly "attached" to the top of the hydrophobic tail chain without any buffer space. This results in a high concentration of positive charge on the surface of the lipid membrane. The excessively high surface charge density causes strong electrostatic repulsion between the lipid molecules of LNPs. This repulsion causes the lipid bilayer, which should be tightly and regularly arranged, to become loose and disordered. In addition, the nebulization process impacts the lipid membrane of LNPs, directly damaging the membrane structure. This ultimately leads to problems such as particle fusion and enlargement, lipid membrane rupture, and mRNA leakage, which completely fails to meet the requirements of nebulized drug delivery. Our proposed solution addresses this technical problem by adjusting the distance from the quaternary ammonium head group to the linker.

[0013] Specifically, the structure of the permanent cationic lipid provided in this solution to improve the stability of atomable lipid nanoparticles is shown in the following formula: .

[0014] In some embodiments, the structure of the permanent cationic lipid provided by this solution for improving the stability of atomizable lipid nanoparticles is shown in the following formula: .

[0015] In some embodiments, the structure of the permanent cationic lipid provided by this solution for improving the stability of atomizable lipid nanoparticles is shown in the following formula: .

[0016] In some embodiments, the structure of the permanent cationic lipid provided by this solution for improving the stability of atomizable lipid nanoparticles is shown in the following formula: .

[0017] In some embodiments, the structure of the permanent cationic lipid provided by this solution for improving the stability of atomizable lipid nanoparticles is shown in the following formula: .

[0018] In some embodiments, the structure of the permanent cationic lipid provided by this solution for improving the stability of atomizable lipid nanoparticles is shown in any of the following formulas:

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026] .

[0027] In some embodiments, this solution provides a structure of a permanently cationic lipid that improves the stability of atomable lipid nanoparticles, as shown in any of the following formulas:

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] .

[0037] In some embodiments, this solution provides a structure of a permanently cationic lipid that improves the stability of atomable lipid nanoparticles, as shown in any of the following formulas:

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] .

[0047] Secondly, this solution provides an application of a permanent cationic lipid that improves the stability of atomizable lipid nanoparticles, for use in the preparation of LNP formulations for atomized drug delivery.

[0048] In some embodiments, ionizable lipids, permanently cationic lipids, auxiliary lipids, cholesterol, and polymeric lipids are mixed and vortexed to obtain a homogeneous and transparent lipid stock solution. mRNA is dissolved in the corresponding buffer solution and vortexed to obtain an aqueous solution. The lipid stock solution and aqueous solution are mixed using a continuous flow microfluidic system to obtain a primary LNP suspension. The primary LNP suspension is purified by dialysis until no organic solvent residue is detected. The LNP formulation is concentrated using an ultrafiltration tube and then filtered under positive pressure through a disposable PES membrane to obtain a sterile LNP product. Thirdly, this solution provides a method for preparing a permanent cationic lipid that improves the stability of atomizable lipid nanoparticles, comprising the following steps: C12~C18 fatty acid propionyl aldehyde and bromoic acid were placed in dichloromethane and stirred for a period of time to obtain a first mixture. Aliphatic isonitriles were added to the first mixture and stirred for a period of time. Then, a THF solution of trimethylamine was added to obtain a second mixture. The second mixture was stirred overnight, the solvent was removed under vacuum, and the residue was purified by rapid column chromatography to obtain a permanent cationic lipid that improves the stability of atomizable lipid nanoparticles.

[0049] In some embodiments, C12-C18 fatty acid propionyl aldehydes are selected from any of the following structural formulas: ; .

[0050] In some embodiments, the bromoacid is selected from one of the following structural formulas: .

[0051] In some embodiments, the aliphatic isonitrile is selected from one of the following structural formulas: .

[0052] In some embodiments, C12-C18 fatty acid propionyl aldehyde and bromoic acid are placed in dichloromethane and stirred for 5-15 min to obtain a first mixture. Aliphatic isonitriles are added to the first mixture and stirred for 10-14 h. Then, a THF solution of trimethylamine is added to obtain a second mixture. The second mixture is stirred overnight, the solvent is removed under vacuum, and the residue is purified by a DCM rapid column of 0-5% MeOH to obtain a permanent cationic lipid that improves the stability of atomizable lipid nanoparticles.

[0053] Compared with existing technologies, this technical solution has the following characteristics and beneficial effects: The permanent cationic lipids (LNPs) provided in this solution, which enhance the stability of atomizable lipid nanoparticles, utilize a flexible linker structure containing ester groups. This effectively increases the distance between the quaternary ammonium positively charged head groups and the hydrophobic framework, dispersing the surface charge density of the lipid membrane. Simultaneously, the ester structure improves the compatibility between lipid molecules and the flexibility of the lipid membrane, ensuring that the prepared LNPs maintain the structural integrity of the lipid bilayer throughout the entire atomization process. After atomization, the LNPs not only maintain a stable average particle size below 200 nm, but also retain a PDI within the acceptable range of ≤0.3, without significant particle aggregation or a significant decrease in mRNA encapsulation efficiency. This fully meets the formulation quality requirements for atomized inhalation drug delivery, overcoming the technical bottleneck of existing permanent cationic lipids being unsuitable for atomized drug delivery scenarios.

[0054] Furthermore, the permanent cationic lipids provided in this solution, which enhance the stability of atomizable lipid nanoparticles, possess pH-insensitive quaternary ammonium salt permanent positive charge centers. These centers can stably maintain positive charge in physiological pH environments, extreme atomization processes, and the respiratory mucosal microenvironment. They can form stable electrostatic interactions with negatively charged sialic acid residues and glycosaminoglycans widely distributed on the respiratory epithelial cells and mucosal surfaces of lung tissue. This significantly enhances the adhesion, accumulation, and long-term retention efficiency of LNPs in the lungs, preventing LNPs from being rapidly cleared by respiratory mucus or entering the systemic circulation, thus avoiding off-target effects and fundamentally improving the lung-targeted delivery efficiency of LNPs. Meanwhile, the excellent atomization stability of this lipid ensures that LNP can still be completely encapsulated and protect mRNA from nuclease degradation after atomization, thus preserving the biological activity of mRNA. It can also form a synergistic effect with the ionizable lipids in the formulation, which can efficiently promote the endocytosis and endosome escape of mRNA, and significantly improve the transfection and protein expression efficiency of mRNA in lung target cells such as alveolar epithelial cells. It truly achieves the simultaneous improvement of lung targeting and delivery transfection efficiency, and solves the industry contradiction that existing lipids improve targeting but transfection efficiency decreases due to structural damage. Attached Figure Description

[0055] Figure 1This is a hydrogen nuclear magnetic resonance image of the permanent cationic lipid prepared in Example 1 of this scheme to improve the stability of atomizable lipid nanoparticles.

[0056] Figure 2 This is a hydrogen nuclear magnetic resonance image of the permanent cationic lipid prepared in Example 2 of this scheme to improve the stability of atomizable lipid nanoparticles.

[0057] Figure 3 This is a hydrogen nuclear magnetic resonance image of the permanent cationic lipid prepared in Example 3 of this scheme to improve the stability of atomizable lipid nanoparticles.

[0058] Figure 4 This is a hydrogen nuclear magnetic resonance image of the permanent cationic lipid prepared in Example 4 of this scheme to improve the stability of atomizable lipid nanoparticles.

[0059] Figure 5 This is a hydrogen nuclear magnetic resonance image of the permanent cationic lipid prepared in Example 5 of this scheme to improve the stability of atomizable lipid nanoparticles.

[0060] Figure 6 This is a graph showing the particle size changes of each LNP before and after atomization.

[0061] Figure 7 This is a schematic diagram of the transfection capability on 293 cells.

[0062] Figure 8 This is a schematic diagram illustrating the transfection capability on A549 cells.

[0063] Figure 9 This is a graph showing the results of the freeze-thaw stability test.

[0064] Figure 10 This is a graph showing the results of the toxicity assessment. Detailed Implementation

[0065] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0066] I. Preparation of permanent cationic lipids: 1) DP-1:

[0067] 1 mmol of 4-bromobutyric acid and 1 mmol of tetradecanoic acid-3-formyl-2,2-dimethylpropyl ester were dissolved in 5 mL of dichloromethane and stirred for 10 minutes. Then, 1 mmol of n-hexadecaneisocyanate was added, and the resulting solution was stirred at room temperature for 12 hours. Then, a THF solution of trimethylamine (2 eq) was added, and the reaction was stirred overnight. The solvent was removed under vacuum, and the target compound was separated by silica gel column chromatography (the elution system was a mixed solution of methanol and dichloromethane, with the methanol ratio ranging from 0% to 5% by volume).

[0068] The test pattern obtained by performing hydrogen nuclear magnetic resonance (NMR) on target compound 1 is shown below. Figure 1 As shown, the corresponding hydrogen nuclear magnetic resonance data are: 1 H NMR (500 MHz, CDCl3, ppm): δ 6.66 (t, 1H), 4.87 (s, 1H), 4.09 (d,1H), 3.80 (d, 1H), 3.70 (m, 2H), 3.29 (m, 11H), 2.69 (m, 1H), 2.56 (m, 1H), 2.31 (t, 2H), 2.12 (m, 2H), 1.94 (m, 2H), 1.60-1.25 (m, 48H), 1.09 (s, 3H), 1.02 (s, 3H), 0.88 (t, 6H).

[0069] 2) DP-N3-1:

[0070] 1 mmol of 3-bromopropionic acid and 1 mmol of tetradecanoic acid-3-formyl-2,2-dimethylpropyl ester were dissolved in 5 mL of dichloromethane and stirred for 10 minutes. Then, 1 mmol of n-hexadecanoic acid was added, and the resulting solution was stirred at room temperature for 12 hours. Then, a THF solution of trimethylamine (2 eq) was added, and the reaction was stirred overnight. The solvent was removed under vacuum, and the solution was separated by silica gel column chromatography (the elution system was a mixed solution of methanol and dichloromethane, with the methanol ratio ranging from 0% to 5% by volume) to obtain the target compound II.

[0071] The test pattern obtained by performing hydrogen nuclear magnetic resonance (NMR) on target compound 2 is shown below. Figure 2 As shown, the corresponding hydrogen nuclear magnetic resonance data are: 1H NMR (500 MHz, CDCl3, ppm): δ 7.61 (t, 1H), 4.92 (s, 1H), 4.23 (d,1H), 4.05 (m, 3H), 3.73 (d, 1H), 3.70 (m, 8H), 2.87 (m, 14H), 2.30 (t, 2H), 1.60-1.25 (m, 40H), 1.14 (s, 3H), 1.06 (s, 3H), 0.88 (t, 6H).

[0072] 3) DP-N2-1: 4)

[0073] 1 mmol of bromoacetic acid and 1 mmol of tetradecanoic acid-3-formyl-2,2-dimethylpropyl ester were dissolved in 5 mL of dichloromethane and stirred for 10 minutes. Then, 1 mmol of n-hexadecaneisocyanate was added, and the resulting solution was stirred at room temperature for 12 hours. Then, a THF solution of trimethylamine (2 eq) was added, and the reaction was stirred overnight. The solvent was removed under vacuum, and the solution was separated by silica gel column chromatography (the elution system was a mixed solution of methanol and dichloromethane, with the methanol ratio ranging from 0% to 5% by volume) to obtain the target compound III.

[0074] The test results of hydrogen nuclear magnetic resonance (NMR) testing of target compound 3 are shown in the figure below. Figure 3 As shown, the corresponding hydrogen nuclear magnetic resonance data are: 1 H NMR (500 MHz, CDCl3, ppm): δ 7.61 (t, 1H), 4.92 (s, 1H), 4.23 (d,1H), 4.05 (m, 3H), 3.73 (d, 1H), 3.70 (m, 8H), 2.87 (m, 14H), 2.30 (t, 2H), 1.60-1.25 (m, 40H), 1.14 (s, 3H), 1.06 (s, 3H), 0.88 (t, 6H).

[0075] 4) DP-sin-1:

[0076] 1 mmol of 4-bromobutyric acid and 1 mmol of tetradecanoic acid-3-formyl-2-methylpropyl ester were dissolved in 5 mL of dichloromethane and stirred for 10 minutes. Then, 1 mmol of n-hexadecanoic acid was added, and the resulting solution was stirred at room temperature for 12 hours. Then, a THF solution of trimethylamine (2 eq) was added, and the reaction was stirred overnight. The solvent was removed under vacuum, and the solution was separated by silica gel column chromatography (the elution system was a mixed solution of methanol and dichloromethane, with the methanol ratio ranging from 0% to 5% by volume) to obtain the target compound four.

[0077] The test results of proton nuclear magnetic resonance (NMR) testing of target compound four are shown in the figure below. Figure 4 As shown, the corresponding hydrogen nuclear magnetic resonance data are: 1 H NMR (500 MHz, CDCl3, ppm): δ 7.65 (s, 1H), 6.49 (d, 1H), 4.97 (s,1H), 4.51 (d, 1H), 4.40 (d, 1H), 3.55 (m, 11H), 3.31 (m, 2H), 2.29 (t, 2H), 1.69 (m, 2H), 1.54 (m, 4H), 1.25 (m, 49H), 1.03 (s, 3H), 0.88 (t, 3H).

[0078] 5) DP-non-1:

[0079] 1 mmol of 4-bromobutyric acid and 1 mmol of tetradecanoic acid-3-formylpropyl ester were dissolved in 5 mL of dichloromethane and stirred for 10 minutes. Then, 1 mmol of n-hexadecanoic acid nitrile was added, and the resulting solution was stirred at room temperature for 12 hours. Then, a THF solution of trimethylamine (2 eq) was added, and the reaction was stirred overnight. The solvent was removed under vacuum, and the solution was separated by silica gel column chromatography (the elution system was a mixed solution of methanol and dichloromethane, with the methanol ratio ranging from 0% to 5% by volume) to obtain the target compound five.

[0080] The test results of hydrogen nuclear magnetic resonance (NMR) testing of target compound five are shown in the figure below. Figure 5 As shown, the corresponding hydrogen nuclear magnetic resonance data are: 1H NMR (500 MHz, CDCl3, ppm): δ 6.83-6.70 (m, 1H), 5.28-5.02 (m, 1H), 4.08 (m, 2H), 3.83 (m, 3H), 3.43 (m, 10H), 3.22 (m, 2H), 2.89 (s, 3H), 2.60(m, 2H), 2.30 (t, 2H), 2.18 (m, 2H), 1.90 (m, 2H), 1.60-1.25 (m, 44H), 1.02-0.95 (dd, 3H), 0.88 (t, 6H).

[0081] II. Preparation of LNP formulations: 1) Take DP-1, DP-N3-1, DP-N2-1, DP-sin-1, DP-non-1, DOTAP and EPC 14:0 as cationic lipids respectively. Mix the ionizable lipids, cationic lipids, auxiliary lipids, cholesterol and polymeric lipids in a molar ratio of 20:20:13:44:3 and vortex to mix them evenly to obtain a homogeneous and transparent lipid mother liquor. Among them, the ionizable lipid is 314BNT (from lipid WO2024141105A1, lipid 424), the polymeric lipid is pep14 (from lipid WO2025168076A1, PL 10-1), and the auxiliary lipid is DSPC.

[0082] 2) Dissolve 0.5 mg / mL mRNA in the corresponding 10 mM sodium acetate buffer (pH 5.5), vortex to mix thoroughly, and stir to obtain an aqueous solution. The concentration of mRNA should be determined according to actual needs.

[0083] 3) The lipid stock solution (organic phase) and the aqueous solution were mixed using a continuous flow microfluidic system. The flow rate ratio of the organic phase to the aqueous phase was set to 1:3 and the total flow rate was 20 mL / min. The mixture was continuously mixed to obtain the initial LNP suspension. 4) The initial LNP suspension was purified by dialysis using a dialysis box with a molecular weight cutoff of 20 kDa and 10 mM Tris buffer (pH 7.4) as the dialysis solution overnight. The volume of the dialysis solution was changed 100 times that of the sample until no organic solvent residue was detected. The LNP preparation was concentrated using a 100 kDa ultrafiltration tube to a concentration of 0.5 mg / mL, and then filtered under positive pressure through a 0.22 μm disposable PES filter membrane to obtain sterile LNP product, which was stored at 4°C for later use.

[0084] Take a small amount of sterile LNP product and test the core indicators. If the following conditions are met: average particle size 50-200 nm, PDI≤0.3, encapsulation rate≥85%, organic solvent residue ≤0.5%, and sterility test qualified, the product can proceed to the atomization experiment stage.

[0085] III. Atomization Experiment: 1) Atomizer parameter adjustment: Confirm the atomization environment temperature is 25℃ and the relative humidity is 50%. Turn on the vibrating screen atomizer and run it unloaded for 5 minutes. Observe the mist output status to ensure that there is no mist interruption or leakage. After the atomization is uniform, stop the unloaded operation and prepare for sample loading.

[0086] 2) Sample loading: In a clean bench, take 1-10 mL of qualified sterile LNP product and slowly inject it into the nebulizer cup. Avoid generating air bubbles during the injection process. Ensure that the sample liquid level is 1-2 cm above the screen surface and does not contact the internal electrodes of the nebulizer to prevent electrode short circuits and sample contamination.

[0087] 2.3 Atomization Operation Seal the nebulizer outlet to the sterile mist collection device (mist collection bottle / collection tank), and place the collection device in a 0-4℃ ice bath environment to reduce the temperature of the nebulized liquid and reduce the degradation of bioactive substances; turn on the nebulizer switch and nebulize continuously for 5-30 minutes, observing the mist output status in real time during the nebulization process.

[0088] 2.4 Sample Collection After nebulization is complete, turn off the nebulizer and collect the nebulized liquid from the collection device; place it in a sterile centrifuge tube, label it, and store it at 4°C. Complete subsequent testing within 24 hours to avoid sample deterioration.

[0089] III. Sample testing after atomization: 3.1 Particle size and PDI detection: The average particle size and PDI of the LNP product before atomization and the combined sample after atomization were detected by DLS method. The detection conditions were 25℃ and scattering angle 173°. The sample was appropriately diluted with sterile 10 mM Tris. The LNP structure was required to be stable after atomization and without obvious aggregation.

[0090] The particle size of each LNP before and after atomization is as follows: Figure 6 As shown, Figure 6 As can be seen, the series of compounds developed in this invention enable LNPs to maintain a stable particle size of less than 200 nm after atomization; in contrast, LNPs using DOTAP or EPC 14:0 exhibit significant particle size changes after atomization, rendering them unusable for subsequent biological function verification. It should be noted that... Figure 6In this context, "DP-1" indicates an LNP prepared using DP-1 as a cationic lipid and bromine as the anion; "DP-sin-1" indicates an LNP prepared using DP-sin-1 as a cationic lipid; "DP-non-1" indicates an LNP prepared using DP-non-1 as a cationic lipid; "DP-1-S3" indicates an LNP prepared using DP-1 as a cationic lipid and iodine as the anion; "DOTAP" indicates an LNP prepared using DOTAP as a cationic lipid; and "EPC 14:0" indicates an LNP prepared using EPC 14:0 as a cationic lipid.

[0091] 3.2 Transfection Capacity Test: LNP formulations were prepared using DP-1 with iodine as the anion, as cationic lipids. Ionizable lipids included 314BNT (from lipid WO2024141105A1, lipid 424), and auxiliary lipids included DSPC, cholesterol, and polymeric lipids pep14 (from lipid WO2025168076A1, PL 10⁻¹) or pep22 (from lipid WO2025168076A1, PL 10⁻²), prepared according to standard methods in a molar ratio of 20:20:13:44:3. LNPs were defined as follows: if pep14 was used, the LNP was named G1; if pep22 was used, the LNP was named G2. The integrity of the nebulized mRNA was then detected by capillary electrophoresis, compared with the integrity of the mRNA before nebulization. Transfection efficiency was also assessed using cell transfection experiments (transfection capacity on 293 cells and A549 cells). Figure 7 and Figure 8 As shown, Figure 7 These are transfection efficiency data on 293FT cells. Figure 8 These are transfection capability data on A549 cells.

[0092] The specific cell transfection experiments are as follows: ① Plated cells: Digest cells according to the culture conditions of different cell types (e.g., 293FT digested with 0.05g trypsin at room temperature for 3 min, A549 digested with 0.25g trypsin at 37°C for 3 min); count cells using a cell counter, centrifuge and discard the supernatant; apply appropriate complete culture medium and adjust the cell density to 53. 10^5 cells / mL; add 200uL PBS to the edge of each well in a 96-well plate to reduce edge effect, and add 100uL cell suspension to each of the remaining wells; incubate at 37°C for 4-6 hours until the cells are fully attached (at which point 70% aggregation can be achieved).

[0093] ② Transfection: Prepare RNA-Lipo as a positive control (see the Lipofectamine™ MessengerMAX™ Transfection Reagent instructions for preparation); dilute all LNP-RNAs with PBS to 100ug RNA / mL; Add 1 μL of LNP-RNA dilution buffer to each well (2 μL is required for special difficult-to-transfect cell lines), and add 3 auxiliary wells for each sample; incubate at 37°C for 24 h.

[0094] ③ Detection: Equilibrate the firefly luciferase reporter gene assay reagent and the test plate to room temperature in advance (equilibration time should not exceed 30 min); add 100 uL of luciferase reporter gene assay reagent to each well of all test wells, and incubate at room temperature in the dark with shaking for 5 min; read the values ​​from the chemiluminescence module of the microplate reader.

[0095] 3.3 Freeze-thaw stability test: The freeze-thaw stability test results of the LNP formulation prepared using DP-1 are as follows: Figure 9 As shown.

[0096] 3.4 Cytotoxicity assessment The LNP formulation prepared using DP-1 was subjected to cytotoxicity assessment on A549 cells using the CCK-8 assay. The cytotoxicity assessment results are as follows: Figure 10 As shown, the LNP has low toxicity, demonstrating its good safety profile.

[0097] Those skilled in the art should understand that the technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0098] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A permanent cationic lipid for improving the stability of atomizable lipid nanoparticles, characterized in that, The general structural formula is as follows: ； Wherein R1 is a C6~C20 alkyl group or an alkenyl group; R2 is a C1~C3 alkyl group; R3 and R4 are hydrogen or C1~C2 alkyl groups; R5 is a C6~C20 alkyl group or contains an alkenyl group; R6 is a C1~C6 alkyl group; R7, R8, and R9 are C1-C3 alkyl groups; X-: Pharmaceutically acceptable anion.

2. The permanent cationic lipid for improving the stability of atomizable lipid nanoparticles according to claim 1, characterized in that, The general structural formula is as follows: 。 3. The permanent cationic lipid for improving the stability of atomizable lipid nanoparticles according to claim 1, characterized in that, The general structural formula is as follows: 。 4. The permanent cationic lipid for improving the stability of atomizable lipid nanoparticles according to claim 1, characterized in that, The general structural formula is as follows: 。 5. The permanent cationic lipid for improving the stability of atomizable lipid nanoparticles according to claim 1, characterized in that, The general structural formula is as follows: 。 6. The permanent cationic lipid for improving the stability of atomizable lipid nanoparticles according to claim 1, characterized in that, The general structural formula is as follows: 。 7. The permanent cationic lipid for improving the stability of atomizable lipid nanoparticles according to claim 1, characterized in that, It is used in the preparation of LNP formulations for nebulized drug delivery.

8. The permanent cationic lipid for improving the stability of atomizable lipid nanoparticles according to claim 7, characterized in that, Ionizable lipids, permanently cationic lipids, auxiliary lipids, cholesterol, and polymeric lipids are mixed and vortexed to obtain a homogeneous and transparent lipid stock solution. mRNA is dissolved in the corresponding buffer solution and vortexed to obtain an aqueous solution. The lipid stock solution and the aqueous solution are mixed using a continuous flow microfluidic system to obtain a primary LNP suspension. The primary LNP suspension is purified by dialysis until no organic solvent residue is detected. The LNP preparation is concentrated using an ultrafiltration tube and then filtered under positive pressure through a disposable PES filter membrane to obtain a sterile LNP product.

9. A method for preparing a permanent cationic lipid to improve the stability of atomizable lipid nanoparticles, characterized in that, Includes the following steps: C12~C18 fatty acid propionyl aldehyde and bromoic acid were placed in dichloromethane and stirred for a period of time to obtain a first mixture. Aliphatic isonitriles were added to the first mixture and stirred for a period of time. Then, a THF solution of trimethylamine was added to obtain a second mixture. The second mixture was stirred overnight, the solvent was removed under vacuum, and the residue was purified by rapid column chromatography to obtain a permanent cationic lipid that improves the stability of atomizable lipid nanoparticles.

10. The method for preparing a permanent cationic lipid for improving the stability of atomizable lipid nanoparticles according to claim 9, characterized in that, C12~C18 fatty acid propionyl aldehydes are selected from any of the following structural formulas: ； ； Bromoacids are selected from one of the following structural formulas: ； Aliphatic isonitriles are selected from one of the following structural formulas: 。

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