Nucleic acid-lipid nanoparticle dry powder inhaler as well as preparation method and application thereof

Abstract

The invention relates to a nucleic acid-lipid nanoparticle dry powder inhaler and a preparation method and application thereof.The inhaler comprises nucleic acid-lipid nanoparticle freeze-dried powder and a dry powder inhalation carrier, and the nucleic acid-lipid nanoparticle freeze-dried powder comprises nucleic acid, lipid, buffer salt and excipient; the lipids include cationic lipids, neutral lipids, auxiliary lipids, and conjugated lipids. The particle size of nucleic acid nanoparticles of the inhalant is controlled to be 115 nm, the encapsulation efficiency is 90% or above, the integrity of nucleic acid is 80% or above, the mass median aerodynamic particle size is 2.53 microns, the effective part deposition rate reaches 60% or above, the cell transfection efficiency is high, and the preparation method is simple and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to a nucleic acid-lipid nanoparticle dry powder inhaler and a preparation method and application thereof. Background Art

[0002] The lungs are the internal organs most susceptible to infection and damage from the external environment, and are frequently exposed to particles, chemicals, and infectious microorganisms in the ambient air. Respiratory diseases impose a huge health burden on the world, and since the COVID-19 pandemic, the number of lung dysfunctions has increased on top of all other lung diseases, and with it comes an inevitable increase in the need for new lung therapies that are applied topically to the site of action.

[0003] RNA therapy is expected to be used to treat respiratory diseases. Compared with traditional treatments using proteins, peptides, small molecules or monoclonal antibodies, RNA therapy offers the possibility of high selectivity, efficacy and personalized treatment. RNA is a negatively charged macromolecule that does not bind to the cell surface or penetrate the cell membrane. In addition, RNA therapy also faces challenges such as nuclease degradation, off-target gene silencing and immunostimulatory effects.

[0004] To overcome these limitations, RNA has been encapsulated in a variety of materials, including lipids, polymers, inorganic materials, proteins, and combinations of the above. Lipid powder aerosol carrier systems that mimic the composition of lung surfactant or cell membranes enhance the ability to overcome the biological barrier of the lungs and reduce toxicity and antigenicity. Although all RNA drugs on the market target the liver, the lungs offer a variety of currently untreatable targets that have the potential to be treated with RNA therapy. Therefore, local pulmonary delivery of RNA nanoparticles may eventually enable delivery beyond the liver. Administration of RNA drugs via dry powder inhalers offers many advantages related to the physical, chemical, and microbiological stability of RNA and nanosuspensions. At the same time, compared with systemic administration, local administration to the lungs has obvious advantages, which can reduce the dose of local action and thus reduce side effects.

[0005] CN116712408A discloses a lipid nanoparticle and a preparation method and application thereof, and provides a lipid powder aerosol carrier comprising: neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids and divalent salts; compared with monovalent salts (sodium acetate), by adopting divalent salts, the transfection efficiency of lipid nanoparticles containing the lipid powder aerosol carrier can be improved, that is, the delivery performance of the lipid nanoparticles is improved; at the same time, the toxicity of the lipid nanoparticles is also reduced.

[0006] In summary, developing a nucleic acid-loaded preparation with excellent quality attributes and applying it to preparations for pulmonary administration to cope with the high incidence of lung diseases and provide more options for clinical medication has become one of the issues that need to be urgently addressed in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a nucleic acid-lipid nanoparticle dry powder inhaler and its preparation method and application. The inhaler provided by the present invention has the following characteristics: 1) the particle size of the nucleic acid nanoparticle is 115.2nm, PDI<0.1, 2) the encapsulation rate is high>90%, 3) the integrity is more than 80%, 4) the mass median aerodynamic particle size is 2.53μm, 5) the effective site deposition rate reaches 60%, 6) the cell transfection efficiency is high. The preparation method of the inhaler provided by the present invention is simple and suitable for industrial production.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a nucleic acid-lipid nanoparticle dry powder inhaler, the inhaler comprising a nucleic acid-lipid nanoparticle lyophilized powder and a powder inhaler carrier, the nucleic acid-lipid nanoparticle lyophilized powder comprising nucleic acid, lipid, buffer salt and excipient, the lipid comprising a cationic lipid, a neutral lipid, an auxiliary lipid and a conjugated lipid;

[0010] The neutral lipid is distearoylphosphatidylcholine (DSPC), the auxiliary lipid is cholesterol, the conjugated lipid is dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), the molar ratio of the cationic lipid, the neutral lipid, the auxiliary lipid and the conjugated lipid is 49:10:39.5:1.5, the buffer salt is Tris-HCl / Tris, the excipient is sucrose, the powder aerosol carrier is mannitol and / or lactose, and the mass ratio of the nucleic acid-lipid nanoparticle lyophilized powder to the powder aerosol carrier is 1:3.

[0011] Preferably, the inhalant further comprises leucine.

[0012] Preferably, the mass ratio of the nucleic acid-lipid nanoparticle lyophilized powder to leucine is 10:(0.5-4), preferably 10:(1-3).

[0013] The specific point values ​​of the above (0.5-4) can be selected from 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4, etc.

[0014] The specific point values ​​of the above (1-3) can be selected as 1, 1.5, 2, 2.5 or 3, etc.

[0015] Preferably, the cationic lipid comprises any one or a combination of at least two of the molecules having the following structures:

[0016]

[0017]

[0018] Preferably, the inhalant is administered by oral inhalation using a mouthpiece.

[0019] Preferably, the pH of the buffer salt is 6 to 8 (eg, 6, 6.5, 7, 7.5 or 8, etc.), preferably pH 7.6.

[0020] Preferably, the particle size D50 of the powder aerosol carrier is 80-120 μm (for example, it may be 80 μm, 90 μm, 100 μm, 110 μm or 120 μm, etc.).

[0021] Preferably, the mass ratio of nucleic acid to lipid is 1:(10-40).

[0022] The specific point values ​​(10-40) mentioned above can be selected from 10, 15, 20, 25, 30, 35 or 40, etc.

[0023] Preferably, the mass ratio of the nucleic acid to the buffer salt is 1:(15-50).

[0024] The specific point values ​​(15-50) mentioned above can be selected from 15, 20, 25, 30, 35, 40, 45 or 50, etc.

[0025] Preferably, the mass ratio of the nucleic acid to the excipient is 1:(300-1000).

[0026] The specific point values ​​of the above (300-1000) can be selected as 300, 400, 500, 600, 700, 800, 900 or 1000, etc.

[0027] Preferably, the nucleic acid-lipid nanoparticle dry powder inhaler comprises a nucleic acid-lipid nanoparticle lyophilized powder and a powder inhaler carrier, the nucleic acid-lipid nanoparticle lyophilized powder comprises nucleic acid, lipids, buffer salts and excipients, the lipids comprise cationic lipids, neutral lipids, auxiliary lipids and conjugated lipids; the cationic lipid is YK-009, the neutral lipid is DSPC, the auxiliary lipid is cholesterol, the conjugated lipid is DMG-PEG2000, the molar ratio of the cationic lipid, neutral lipid, auxiliary lipid and conjugated lipid is 49:10:39.5:1.5, the buffer salt is Tris-HCl / Tris, the excipient is sucrose, the powder inhaler carrier is mannitol and / or lactose, and the mass ratio of the nucleic acid-lipid nanoparticle lyophilized powder to the powder inhaler carrier is 1:3.

[0028] Preferably, the nucleic acid comprises deoxyribonucleic acid and / or ribonucleic acid.

[0029] Preferably, the ribonucleic acid comprises any one or a combination of at least two of small interfering RNA, asymmetric interfering RNA, micro RNA, Dicer-substrate RNA, small hairpin RNA, messenger RNA, long non-coding RNA, circular RNA, self-amplifying RNA or 5'-pppRNA.

[0030] Preferably, the nucleotides of the nucleic acid are any one of eGFP mRNA, the sequence as shown in SEQ ID NO.1, or the sequence as shown in SEQ ID NO.2.

[0031] In a second aspect, the present invention provides a method for preparing the inhalant according to the first aspect, the preparation method comprising the following steps:

[0032] (1) preparing an aqueous phase containing nucleic acid and an organic phase containing lipid, and encapsulating the nucleic acid using microfluidics to obtain a nucleic acid-lipid nanoparticle solution;

[0033] (2) dialyzing the nucleic acid-lipid nanoparticle solution in a buffer solution;

[0034] (3) adding a freeze-drying protective agent;

[0035] (4) freeze-drying the solution obtained in step (3) to obtain a freeze-dried powder;

[0036] (5) The freeze-dried powder is pre-crushed or not crushed and mixed with a powder inhaler carrier to obtain a dry powder inhaler.

[0037] Preferably, the preparation method specifically comprises the following steps:

[0038] (a) Water phase preparation:

[0039] The mRNA stock solution was dissolved in pH 4.0 citrate buffer to prepare the aqueous phase.

[0040] (b) Organic phase preparation:

[0041] Cationic lipid, neutral lipid, auxiliary lipid and conjugated lipid were dissolved in anhydrous ethanol at a molar ratio of 49:10:39.5:1.5 to prepare an organic phase.

[0042] (c) Encapsulation:

[0043] The organic phase and the aqueous phase are quickly mixed in a microfluidic device for encapsulation. The encapsulation method is that the ratio of the organic phase and the aqueous phase inflow rate is 1:(2-4), and the total inflow flow rate is 8-20 mL / min.

[0044] (d)Dialysis:

[0045] After encapsulation, the sample was placed in a dialysis bag and dialyzed overnight in a pH 6-8 buffer to obtain an mRNA lipid nanoparticle (mRNA-LNP) sample.

[0046] (e) Preparation of freeze-drying protective agent:

[0047] The excipients were dissolved in nuclease-free water to prepare the freeze-drying protectant solution.

[0048] (f) Preparation of mRNA-LNP freeze-dried preparation using freeze-drying protective agent

[0049] The freeze-drying protective agent solution prepared in step (e) is added to the mRNA-LNP sample in step (d) to obtain a mixed solution to be freeze-dried, so that the mass concentration of the excipient in the mixed solution to be freeze-dried is 5-15%, and the solution is freeze-dried to control the water content to be less than 1.0% to obtain nucleic acid-lipid nanoparticle freeze-dried powder.

[0050] (g) Mixing of nucleic acid-lipid nanoparticle freeze-dried powder and powder aerosol carrier

[0051] The nucleic acid-lipid nanoparticle freeze-dried powder and the powder inhaler carrier are mixed at a mass ratio of 1:3, and the mixing is stirred at 100-200 rpm and a shear rate of 1500-2500 rpm. After mixing, a nucleic acid-lipid nanoparticle dry powder inhaler is obtained.

[0052] Preferably, step (5) further comprises the step of mixing with leucine.

[0053] Preferably, after freeze-drying in step (4), the moisture content is ≤1.0% (for example, it may be 0.2%, 0.4%, 0.6%, 0.8% or 1.0%, etc.).

[0054] In a third aspect, the present invention provides use of the inhalant as described in the first aspect in the preparation of nucleic acid drugs.

[0055] In a fourth aspect, the present invention provides use of the inhalant as described in the first aspect in the preparation of a medicament for treating a disease or condition in a mammal.

[0056] Preferably, the disease or condition comprises any one of an infectious disease, a proliferative disease, a genetic disease, an autoimmune disease, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease or a metabolic disease.

[0057] Preferably, the disease is cancer.

[0058] Preferably, the disease is diabetes.

[0059] Preferably, the infectious disease includes any one of a disease caused by coronavirus, a disease caused by influenza virus, a disease caused by HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies or herpes.

[0060] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] The invention provides a nucleic acid-lipid nanoparticle dry powder inhaler. The inhaler comprises nucleic acid-lipid nanoparticle freeze-dried powder and a powder aerosol carrier. The nucleic acid-lipid nanoparticle freeze-dried powder comprises nucleic acid, lipid, buffer salt and excipient. The lipid comprises YK-009, DSPC, cholesterol and DMG-PEG2000, the molar ratio of the four lipids is 49:10:39.5:1.5, the buffer salt is Tris-HCl / Tris, the excipient is sucrose, the powder aerosol carrier is mannitol, and the mass ratio of the nucleic acid-lipid nanoparticle freeze-dried powder to the powder aerosol carrier is 1:3. The present invention forms a whole through the interaction of specific excipients, specific carriers and specific nucleic acid lipid nanoparticles, achieving a significant technical effect of improving the effective site deposition rate and encapsulation rate, and has the following characteristics: 1) the particle size of the nucleic acid nanoparticles is 115.2nm, PDI <0.1, 2) the encapsulation rate is high> 90%, 3) the integrity is more than 80%, 4) the mass median aerodynamic particle size is 2.53μm, 5) the effective site deposition rate reaches 60%, 6) the cell transfection efficiency is obvious. Specifically:

[0063] (1) Pulverization investigation: Whether the nucleic acid-lipid nanoparticle freeze-dried powder is pre-pulverized or not, the formulation characteristics of the dry powder inhaler are not much different. The particle size is 115.2nm-123.5nm, the encapsulation efficiency is 92.2%-90.1%, the integrity is 83.1%-81.3%, the mass median aerodynamic particle size is 2.53μm-2.76μm, and the effective site deposition rate is 60.6%-64.3%, all of which perform well.

[0064] (2) Types of buffer solutions and pH range: pH 7.0-8.0 Tris-HCl / Tris and pH 7.6 DPBS had good effects, with small particle sizes of 115.2nm-123.5nm and uniform distribution, high encapsulation efficiency of 90.1%-92.2%, high integrity of 81.3%-83.1%, mass median aerodynamic particle size of 2.53μm-3.65μm, and effective site deposition rate of 64.3%-52.4%. pH 7.6 phosphate buffer solution had a slightly worse effect, with a particle size of 129.6nm, an encapsulation efficiency of 86.8%, an integrity of 78.4%, a mass median aerodynamic particle size of 4.17μm, and an effective site deposition rate of 48.2%.

[0065] (3) Types of excipients: Sucrose, trehalose and maltose can all be used as excipients, among which sucrose and trehalose have better effects. Specifically, after reconstitution with sucrose and trehalose, the particle size of lipid nanoparticles was 115.2nm~127.3nm, PDI was <0.1, the particle size distribution was uniform, the encapsulation rate was 91.2%~92.2%, the integrity was 82.1%~83.1%, the aerodynamic mass median was 2.53μm~2.85μm, all <5μm, and the effective site deposition rate was 55.9%~64.3%; the particle size of maltose was 154.6nm, the encapsulation rate was 82.4%, the integrity was 80.7%, the mass median aerodynamic particle size was 3.97μm, and the effective site deposition rate was 50.3%.

[0066] (4) Carrier type: Mannitol, lactose, chitosan, xylitol and hyaluronic acid can all be used as carriers to deliver nucleic acid lipid nanoparticles. The formulation properties are good, and mannitol and lactose have better effects on mass median aerodynamic particle size and effective site deposition rate. Specifically, after reconstitution, the particle size of lipid nanoparticles is 115.2nm~127.4nm, PDI is <0.1 and the distribution is uniform, the encapsulation rate is 90.4%~92.2%, the integrity is 80.9%~83.1%, the aerodynamic mass median is 2.53μm~4.63μm, all <5μm, and the effective site deposition rate is 48.7%~64.3%. The carrier is mannitol, the aerodynamic mass median is 2.53μm, and the effective site deposition rate is 64.3%, respectively, which is more effective.

[0067] (5) Carrier dosage: The mass ratio of freeze-dried powder to carrier is 1:3, 1:2, and 1:4, which can all be used to deliver nucleic acid lipid nanoparticles. The formulation characteristics are all good. The mass ratio of the median aerodynamic particle size and the effective site deposition rate is 1:3, which is more effective. Specifically, after reconstitution, the particle size of the lipid nanoparticles is 115.2nm~120.4nm, PDI<0.1, the encapsulation rate is 90.4%~92.2%, the integrity is 80.3%~83.1%, the median aerodynamic mass is 2.53μm~3.76μm, all <5μm, and the effective site deposition rate is 52.2%~64.3%. Among them, the mass ratio of 1:3 has a median aerodynamic mass of 2.53μm and a deposition rate of 64.3% in the effective site, which is more effective.

[0068] (6) Leucine dosage: The addition of leucine significantly improved the formulation characteristics, mass median aerodynamic particle size and effective site deposition rate. The mass ratio of lyophilized powder to leucine was 10:0.5 to 10:3, and the effect was better. The leucine addition ratio was 10:0.5 to 10:3, the aerodynamic mass median diameter was 2.06 μm to 2.37 μm, and the effective site deposition rate was 65.7% to 67.7%, and the effect was better.

[0069] (7) Different lipid compositions: Dry powder inhalers prepared using different cationic lipids SM-102, ALC-0315, D-Lin-MC3-DMA, YK-401, YK-305 and DOTAP can also be delivered. Among them, SM-102, YK-401, YK-305 and DOTAP as cationic lipids have better particle size distribution, encapsulation efficiency, integrity, mass median aerodynamic particle size and effective site deposition rate than ALC-0315 and D-Lin-MC3-DMA. The transfection effect shows that SM-102, YK-401, YK-305 are consistent with YK-009, while ALC-0315, D-Lin-MC3-DMA and DOTAP are slightly worse than YK-009. The particle size distribution, encapsulation efficiency, integrity, mass median aerodynamic particle size and effective site deposition rate of dry powder inhalers prepared by replacing DSPC with different neutral lipids DOPE and DPPC, and replacing DMG-PEG2000 with conjugated lipid ALC-0159 were slightly worse. The transfection effect showed that the effects of replacing neutral lipids and conjugated lipids were slightly worse. DETAILED DESCRIPTION

[0070] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0071] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0072] Glossary:

[0073] Effective site deposition rate: The effective site is the in vitro simulated inhalation administration. The powder of the preparation is sieved after inhalation, and the content of effective ingredients at different pore sizes is counted. Among them, particles below 4.7μm are deliverable to the lungs. Therefore, the effective site deposition rate refers to the mass percentage of drugs contained in particles below 4.7μm in size in the total delivered dose.

[0074] DSPC: distearoylphosphatidylcholine;

[0075] DMG-PEG2000: dimyristoylglycerol-3-methoxypolyethylene glycol 2000;

[0076] The experimental data of this application were statistically analyzed between groups using the t-test program in EXCEL software, and the P value was calculated. The difference was considered statistically significant when P < 0.05.

[0077] Preparation Example 1

[0078] This preparation example provides a method for preparing a nucleic acid-lipid nanoparticle dry powder inhaler, the method comprising the following steps:

[0079] (1) Water phase preparation:

[0080] The mRNA stock solution was dissolved in pH 4.0 citrate buffer to prepare the aqueous phase.

[0081] (2) Organic phase preparation:

[0082] The cationic lipid, the neutral lipid, the auxiliary lipid and the conjugated lipid are dissolved in anhydrous ethanol to prepare an organic phase.

[0083] (3) Encapsulation:

[0084] The organic phase and the aqueous phase were quickly mixed in a microfluidic device (manufacturer: Precision NanoSystems) for encapsulation. The encapsulation method was that the ratio of the organic phase and the aqueous phase inflow rate was 1:3, the total inflow flow rate was 12 mL / min, and the chip model was Ignite NxGen.

[0085] (4) Dialysis:

[0086] After encapsulation, the sample was placed in a dialysis bag (purchased from Shanghai Yuanye, 50KD) and dialyzed overnight in a 25 mM buffer with a pH value of 7.6 to obtain an mRNA lipid nanoparticle (mRNA-LNP) sample.

[0087] (5) Preparation of freeze-drying protective agent:

[0088] Dissolve sucrose in nuclease-free water to prepare a freeze-drying protectant solution.

[0089] (6) Preparation of mRNA-LNP freeze-dried preparations using freeze-drying protective agents

[0090] The freeze-drying protective agent solution prepared in step (5) is added to the mRNA-LNP sample in step (4) to obtain a mixed solution to be freeze-dried, and the solution is freeze-dried to control the moisture content to less than 1.0% to obtain nucleic acid-lipid nanoparticle freeze-dried powder.

[0091] (7) Mixing of nucleic acid-lipid nanoparticle freeze-dried powder and powder aerosol carrier

[0092] The above-mentioned nucleic acid-lipid nanoparticle freeze-dried powder and mannitol (Pearlitol 100SD, D50: 113 μm, purchased from Merck, Germany) were mixed, and the mixing was stirred at 150 rpm and the shear rate was 2000 rpm. After the mixing was completed, a nucleic acid-lipid nanoparticle dry powder inhaler was obtained.

[0093] Example 1

[0094] This embodiment provides a nucleic acid-lipid nanoparticle dry powder inhaler, which is prepared according to the method provided in Preparation Example 1. The composition of the nucleic acid-lipid nanoparticle dry powder inhaler is shown in Table 1. The molar ratio of YK-009 (prepared with reference to CN114044741A), DSPC (purchased from Nippon Seika Co., Ltd.), cholesterol (purchased from Nippon Seika Co., Ltd.) and DMG-PEG2000 (purchased from Xiamen Sinobond Biotechnology Co., Ltd.) is 49:10:39.5:1.5, the mass ratio of nucleic acid to lipid is 1:17, the mass ratio of nucleic acid to buffer salt is 1:36, the mass ratio of nucleic acid to excipient is 1:1000, and the mass ratio of nucleic acid-lipid nanoparticle lyophilized powder to mannitol is 1:3.

[0095] Table 1

[0096]

[0097]

[0098] Example 2

[0099] This embodiment provides a nucleic acid-lipid nanoparticle dry powder inhaler, which is prepared by referring to the method provided in Preparation Example 1. The composition of the nucleic acid-lipid nanoparticle dry powder inhaler is shown in Table 2. The molar ratio of SM-102, DSPC, cholesterol and DMG-PEG2000 is 49:10:39.5:1.5, the mass ratio of nucleic acid to lipid is 1:20, the mass ratio of nucleic acid to buffer salt is 1:30, the mass ratio of nucleic acid to excipient is 1:900, and the mass ratio of nucleic acid-lipid nanoparticle lyophilized powder and lactose is 1:3.

[0100] Table 2

[0101]

[0102] Example 3

[0103] The present embodiment provides a nucleic acid-lipid nanoparticle dry powder inhaler, which is prepared by referring to the method provided in Preparation Example 1. The composition of the nucleic acid-lipid nanoparticle dry powder inhaler is shown in Table 3. The molar ratio of YK-401, DSPC, cholesterol and DMG-PEG2000 is 49:10:39.5:1.5, the mass ratio of nucleic acid to lipid is 1:10, the mass ratio of nucleic acid to buffer salt is 1:40, the mass ratio of nucleic acid to excipient is 1:1000, and the mass ratio of nucleic acid-lipid nanoparticle lyophilized powder to mannitol is 1:3.

[0104] Table 3

[0105]

[0106]

[0107] Example 4

[0108] This example prepares a nucleic acid-lipid nanoparticle dry powder inhaler, which is different from Example 1 only in that when using the preparation method provided in Preparation Example 1, the nucleic acid-lipid nanoparticle freeze-dried powder obtained in step (6) is crushed by an airflow mill, and the D90 is controlled to be 1.0-5.0 μm to obtain a nucleic acid-lipid nanoparticle freeze-dried powder. The freeze-dried powder is mixed with mannitol in step (7) at a mass ratio of 1:3, and the mixing parameters are the same as those in Preparation Example 1 to obtain a nucleic acid-lipid nanoparticle dry powder inhaler.

[0109] Example 5

[0110] This example prepares a nucleic acid-lipid nanoparticle dry powder inhaler, which differs from Example 1 only in that the pH 7.6 Tris-HCl / Tris buffer is replaced with the buffer in Table 4

[0111] Table 4

[0112] Group Buffer Type Example 5-1 pH 7.6 DPBS buffer solution Example 5-2 pH 7.6 phosphate buffer solution Example 5-3 pH 7.0 Tris-HCl / Tris buffer solution Example 5-4 pH 8.0 Tris-HCl / Tris buffer solution

[0113] Example 6

[0114] This example prepares a nucleic acid-lipid nanoparticle dry powder inhaler, which is different from Example 1 only in that the excipient sucrose is replaced with the excipients in Table 5.

[0115] Table 5

[0116] Group Type of excipients Example 6-1 Trehalose Example 6-2 maltose

[0117] Example 7

[0118] This example prepares a nucleic acid-lipid nanoparticle dry powder inhaler, which is different from Example 1 only in that the powder inhaler carrier mannose is replaced with the powder inhaler carrier in Table 6.

[0119] Table 6

[0120] Group Type of excipients D50(μm) source Example 7-1 lactose 106 German DFE Example 7-2 Chitosan 95 Hunan Xinlvfang Pharmaceutical Co., Ltd. Example 7-3 Xylitol 108 Shaanxi Panlong Yihai Pharmaceutical Co., Ltd. Example 7-4 Hyaluronic acid 112 Avituo

[0121] Example 8

[0122] This example prepares a nucleic acid-lipid nanoparticle dry powder inhaler, which is different from Example 1 only in that the mass ratio of nucleic acid-lipid nanoparticle lyophilized powder to mannitol is replaced by the mass ratio in Table 7.

[0123] Table 7

[0124] Group Quality Ratio Example 8-1 1:2 Example 8-2 1:4

[0125] Example 9

[0126] This example prepares a nucleic acid-lipid nanoparticle dry powder inhaler, which is different from Example 1 only in that when using the preparation method provided in Preparation Example 1, step (7) is to mix the nucleic acid-lipid nanoparticle lyophilized powder, mannitol and leucine, and the mixing is stirred at 150 rpm and the shear rate is 2000 rpm. After mixing, a nucleic acid-lipid nanoparticle dry powder inhaler is obtained, and the mass ratio of the nucleic acid-lipid nanoparticle lyophilized powder to mannitol is 1:3, and the mass ratio of the nucleic acid-lipid nanoparticle lyophilized powder to leucine is shown in Table 8.

[0127] Table 8

[0128] Group The mass ratio of nucleic acid-lipid nanoparticle lyophilized powder to Example 9-1 10:0.5 Example 9-2 10:1 Example 9-3 10:3 Example 9-4 10:4

[0129] Example 10

[0130] This example prepares a nucleic acid-lipid nanoparticle dry powder inhaler, which is different from Example 1 only in that the lipids are replaced with the lipids in Table 9.

[0131] Table 9

[0132]

[0133]

[0134] Comparative Example 1

[0135] In this comparative example, a nucleic acid-lipid nanoparticle dry powder inhaler is prepared. The only difference from Example 1 is that when using the preparation method provided in Preparation Example 1, 1 g of mannitol is dissolved in 46 mL of water to obtain an aqueous solution of the excipient, which is fully dissolved in the mRNA-LNP obtained in step (4) and then spray-freeze-dried to prepare a lyophilized powder, and the lyophilized powder is mixed with mannitol in a mass ratio of 1:3.

[0136] Comparative Example 2

[0137] This comparative example prepares a nucleic acid-lipid nanoparticle dry powder inhaler, which is different from Example 1 only in that when using the preparation method provided in Preparation Example 1, the mRNA-LNP obtained in step (4) is mixed with 0.5% poloxamer 188 and atomized using a vibrating mesh atomizer.

[0138] Test Example 1

[0139] This test example tests the formulation characteristics of the nucleic acid-lipid nanoparticle dry powder inhaler prepared in Examples 1-10 and Comparative Examples 1-2, and the test indicators and methods are as follows.

[0140] (1) Particle size and particle size dispersion index (PDI):

[0141] The particle size and PDI were detected by dynamic light scattering (DLS) at 25±1°C in a nano-laser particle size analyzer (manufacturer: MALVERN; model: ZSU3305). 60 μL of the sample was taken and placed in a quartz micro-sample cell for detection. The detection parameter settings are shown in Table 10.

[0142] Table 10

[0143] temperature 25℃ Equilibrium time 30s Material Liposomes Dispersed System 10% sucrose Detection Mode automatic

[0144] Particle size and PDI are important features to characterize liposomes. There is a liposome fusion process during the freeze-thaw process. Small-sized liposomes fuse into liposomes with slightly larger particle sizes. As the number of freeze-thaw cycles increases, the liposome particle size will increase. Within a certain range, the smaller the particle size, the smaller the change in particle size after freeze-drying, indicating that the product quality and process are better. The dispersion coefficient is the distribution of particle size. The smaller the dispersion coefficient, the more uniform the liposome particle size.

[0145] (2) Encapsulation efficiency:

[0146] The sample was diluted to 2.8 μg / mL, and a portion was mixed with an equal volume (50 μL) of Triton X-100 to fully break the emulsion for determination of total mRNA concentration, and the other portion was mixed with an equal volume (50 μL) of TE (Tris-EDTA) for determination of free unencapsulated mRNA concentration. After the samples with TE and Triton X-100 were incubated at 37°C for a certain period of time, 100 μL of Ribogreen reagent diluted 200 times was added, and after centrifugation to remove bubbles, a multifunctional microplate reader was used to measure the fluorescence value at an excitation wavelength of 485 nm and an emission wavelength of 528 nm, and the plate was read.

[0147] (3) Integrity:

[0148] The mRNA in the mRNA-LNP was extracted using a kit, denatured at 65°C after dilution, and placed on ice after the test sample was prepared. In addition, the gel buffer was prepared, and the prepared test solution, gel buffer, and RNase-free water were added to the sample plate in sequence, and the sample mRNA integrity spectrum was determined using the PA800 Plus gel electrophoresis principle.

[0149] (4) Mass median aerodynamic particle size and effective site deposition rate:

[0150] The Andersen disc impactor ACI was used to detect the drug distribution deposition during drug administration at an airflow rate of 30L / min, and the effective site deposition rate (the active ingredient contained in particles smaller than 4.7μm) and the aerodynamic mass median diameter were calculated. The test method is as follows:

[0151] ① Connect the equipment, turn on the vacuum pump connected to the impactor ACI, and close the two-way solenoid valve.

[0152] ② Take a dose of dry powder inhaler (labeled amount is 100μg) and place it in the mouthpiece adapter.

[0153] ③Open the two-way solenoid valve for 8 seconds to suck the powder into the impactor. After the powder is sucked, wait for 8 seconds and turn off the vacuum pump.

[0154] ④ Use water to extract the drugs in the nozzle adapter, pre-separator, L-shaped connecting tube, the inner wall of each layer and the collection plate.

[0155] ⑤ Use water to extract the drugs on the inner wall of each layer and the corresponding collection plate.

[0156] ⑥ Determine the amount of drug in the above solution.

[0157] According to the amount of drug in the above solution, a cumulative deposition rate and particle size curve was drawn.

[0158] From the above curve we can get:

[0159] (I) Mass median aerodynamic diameter: When the total mass of particles of various sizes smaller than a certain aerodynamic diameter accounts for 50% of the total mass of particles (i.e. the sum of the masses of all particles of different sizes), this diameter is called the mass median diameter. In other words, half of the particles with this median diameter have a particle size smaller than this diameter, and half have a particle size larger than this diameter.

[0160] (II) Deposition rate at effective site: the mass percentage of drug with particle size below 4.7 μm in the total delivered dose.

[0161] The nucleic acid-lipid nanoparticles provided in Example 1-10 and Comparative Example 1-2 were used to encapsulate eGFP mRNA (purchased from GenScript Biotech). The test results are shown in Table 11.

[0162] Table 11

[0163]

[0164]

[0165] Comparative Example 2*: Particle size, encapsulation efficiency and integrity are data after atomization.

[0166] The nucleic acid-lipid nanoparticles provided in Example 1 and Example 10 were used to encapsulate the mRNA shown in SEQ ID NO. 1. The test results are shown in Table 12.

[0167] Table 12

[0168]

[0169] The nucleic acid-lipid nanoparticles provided in Example 1 and Example 10 were used to encapsulate the mRNA shown in SEQ ID NO. 2. The test results are shown in Table 13.

[0170] Table 13

[0171]

[0172] The following conclusions can be drawn from Table 11, Table 12 and Table 13:

[0173] (1) Comparing Example 1 with Example 4, it can be seen that the difference in particle size distribution of the nucleic acid-lipid nanoparticle lyophilized powder is small whether it is pre-crushed or not; the encapsulation rate of the nucleic acid-lipid nanoparticle lyophilized powder that is pre-crushed is slightly lower than that of the uncrushed one, but the decline is small, and the encapsulation rate is still>90%; the integrity of the nucleic acid-lipid nanoparticle lyophilized powder that is pre-crushed is slightly lower than that of the uncrushed one, but the decline is small, and the integrity is still>80%; whether the nucleic acid-lipid nanoparticle lyophilized powder is pre-crushed or not, the mass median aerodynamic particle size is 2.53μm~2.76μm and is <5μm, the effective site deposition rate is 60.6%~64.3%, and the delivery effect is not much different.

[0174] (2) Comparing Example 1 with Example 5, it can be seen that the dry powder inhalers prepared by using different types of buffer solutions and Tris-HCl / Tris buffer solutions with different pH values, the lipid nanoparticles after reconstitution have a particle size of <130 nm, a PDI of <0.1, and a uniform particle size distribution. In Example 1, the smallest particle size is 115.2 nm when dialyzed with pH7.6 Tris-HCl / Tris buffer solution, followed by pH7.6 DPBS buffer solution, and the largest particle size when dialyzed with pH7.6 phosphate buffer solution. When dialyzed with Tris-HCl / Tris buffer solutions with different pH values, the particle size changes slightly within the pH range of 7.0 to 8.0.

[0175] Dry powder inhalers were prepared using different types of buffer solutions. The encapsulation rates of pH 7.6 Tris-HCl / Tris buffer and pH 7.6 DPBS buffer were >90%, and the mRNA encapsulation effect was good. The encapsulation rate of pH 7.6 phosphate buffer solution was relatively low. The encapsulation rate was high when dialyzed using pH 7.0-8.0 Tris-HCl / Tris buffer.

[0176] The integrity of the dialyzed solution was >80% in pH 7.0-8.0 Tris-HCl / Tris buffer and pH 7.6 DPBS buffer, and the integrity of the dialyzed solution was slightly lower in pH 7.6 phosphate buffer.

[0177] The mass median aerodynamic particle size showed that the effect of pH 7.6 phosphate buffer solution was slightly worse than that of pH 7.6 Tris-HCl / Tris buffer and pH 7.6 DPBS buffer. The deposition rate at effective sites was best with pH 7.0-8.0 Tris-HCl / Tris buffer, followed by pH 7.6 DPBS buffer, and pH 7.6 phosphate buffer solution was the worst.

[0178] (3) Comparing Example 1 with Example 6, it can be seen that sucrose, trehalose and maltose are used as excipients respectively. The particle size of the lipid nanoparticles after reconstitution is larger when maltose is used, indicating that the excipients are selected from sucrose and trehalose, and the particle size is uniform. The particle size of the sample prepared by maltose is slightly larger.

[0179] Sucrose and trehalose were used as excipients, respectively, with an encapsulation rate of >90%, and maltose had a slightly lower encapsulation rate of 82.4%, indicating that the excipients selected from the above three types all had good mRNA encapsulation effects, among which sucrose and mannitol were more effective.

[0180] Sucrose, trehalose and maltose were used as excipients respectively, and the differences in the integrity of mRNA were small, but all were relatively high, indicating that when the excipients were selected from the above three types, the mRNA integrity was good.

[0181] The median aerodynamic masses were 2.53μm, 2.85μm, and 3.96μm, all <5μm, and the effective site deposition rates were 64.3%, 55.9%, and 50.3%, respectively. This indicates that the excipients selected from the above three types have good mass median aerodynamic particle size and effective site deposition rate, and can all be used for delivery, among which sucrose and trehalose are more effective.

[0182] (4) By comparing Example 1 with Example 7, it can be seen that mannitol, lactose, chitosan, xylitol and hyaluronic acid are used as carriers respectively. After reconstitution, the particle size of the lipid nanoparticles is <130 nm, ranging from 115.2 nm to 127.4 nm, and the PDI is <0.1. The particle size distribution is uniform, indicating that the carriers are selected from the above five types, and the particle size is uniform and well distributed.

[0183] Mannitol, lactose, chitosan, xylitol and hyaluronic acid were used as carriers respectively, and the encapsulation rates were between 90.4% and 92.2%, all greater than 90%, indicating that the five carriers selected from the above all had good mRNA encapsulation effects.

[0184] Mannitol, lactose, chitosan, xylitol and hyaluronic acid were used as carriers respectively, and the integrity of mRNA was between 80.9% and 83.1%, with little difference in integrity, which was relatively high. This shows that the carriers were selected from the above five types and the mRNA integrity was good.

[0185] The median aerodynamic mass is 2.53μm to 4.63μm, all <5μm, and the effective site deposition rate is 48.7% to 64.3%, indicating that the carriers selected from the above five can be delivered. Among them, the carriers of Example 1 and Example 7-1 are mannitol and lactose, with aerodynamic mass medians of 2.53μm and 2.67μm, respectively, and effective site deposition rates of 64.3% and 59.4%, respectively, which are more effective.

[0186] (5) By comparing Example 1 with Example 8, it can be seen that when mannitol is used as a carrier, the mass ratio of the lyophilized powder to the carrier is 1:3, 1:2, and 1:4. After reconstitution, the particle size of the lipid nanoparticles is slightly different and the distribution is uniform, indicating that the mass ratio of the lyophilized powder to the carrier is between 1:2 and 1:4, and the particle size is uniform and well distributed.

[0187] When mannitol was used as a carrier, the mass ratio of lyophilized powder to carrier was 1:3, 1:2, and 1:4, and the encapsulation efficiency was greater than 90%, indicating that the mass ratio of lyophilized powder to carrier was 1:2 to 1:4, and the encapsulation effect on mRNA was good.

[0188] When mannitol was used as a carrier, the mass ratio of lyophilized powder to carrier was 1:3, 1:2, and 1:4. The difference in mRNA integrity was small, all >80%, indicating that the mRNA integrity was good when the mass ratio of lyophilized powder to carrier was 1:2 to 1:4.

[0189] The median aerodynamic mass was between 2.53 μm and 3.76 μm, all <5 μm, and the deposition rate at the effective site was between 52.2% and 60.3%, indicating that the mass ratio of freeze-dried powder to carrier was between 1:2 and 1:4, which could be delivered. Among them, the median aerodynamic mass was 2.53 μm when the mass ratio was 1:3, and the deposition rate at the effective site was 64.3%, which was more effective.

[0190] (6) Comparing Example 1 with Example 9, it can be seen that the particle size after reconstitution in Example 1 without adding leucine is 115.2 nm, and in Example 9, leucine is added, wherein the mass ratios of nucleic acid-lipid nanoparticle lyophilized powder to leucine are 10:0.5, 10:1, 10:3, and 10:4, respectively. After reconstitution, the particle size of the lipid nanoparticles is 110.6 nm to 116.4 nm, and the PDI is <0.1, indicating that the addition of leucine helps to reduce the particle size. The mass ratio of lyophilized powder to leucine is 10:0.5 to 10:4, and the particle size distribution is good.

[0191] The encapsulation efficiency is between 93.0% and 93.3%, with small differences, all of which are relatively high, indicating that the encapsulation efficiency is good when the ratio is within the range of 10:0.5 to 10:4.

[0192] The integrity is between 84.0% and 84.4%, with small differences, all of which are relatively high, indicating that the integrity is good when the addition ratio is within the range of 10:0.5 to 10:4.

[0193] The aerodynamic mass median diameter was between 2.06 μm and 2.53 μm, all less than 5 μm, and the deposition rate at the effective site was between 64.2% and 67.7%, indicating that the mass ratio of freeze-dried powder to leucine was between 10:0.5 and 10:4, which could be delivered. The aerodynamic mass median diameter tended to decrease and the deposition rate at the effective site tended to increase significantly when the addition ratio was between 10:0.5 and 10:3.

[0194] (7) Comparing Example 1 with Example 10, it can be seen that different lipid compositions are used. Examples 10-1 to 10-6 use different cationic lipids to replace YK-009. There are certain differences in the particle size of lipid nanoparticles after reconstitution. Among them, the cationic lipids ALC-0315 and D-Lin-MC3-DMA have a significantly larger particle size than other examples, followed by DOTAP, and SM-102, YK-401 and YK-305 are close to YK-009 in Example 1. Examples 10-7 and 10-8 use different neutral lipids to replace DSPC, and Example 10-9 uses the conjugated lipid ALC-0159 to replace DMG-PEG2000. The particle size effect is worse than that of Example 1.

[0195] In Examples 10-1 to 10-6, different cationic lipids were used to replace YK-009, among which ALC-0315, D-Lin-MC3-DMA and DOTAP had an encapsulation efficiency of <90%, and SM-102, YK-401 and YK-305 were close to YK-009 and all >90%. In Examples 10-7 and 10-8, different neutral lipids were used to replace DSPC, and in Example 10-9, the conjugated lipid ALC-0159 was used to replace DMG-PEG2000, and the encapsulation efficiency was <90%, which was lower than that in Example 1.

[0196] Using different lipid compositions, the integrity of Examples 10-2, 10-3, 10-7 to 10-9 is slightly lower than that of other lipids, indicating that cationic lipids using SM-102, YK-401, YK-305 and DOTAP are effective, while replacing neutral lipids and conjugated lipids is not as effective as the lipid composition of Example 1.

[0197] Using different lipid compositions, the mass median aerodynamic particle size is 2.53μm to 4.88μm, all <5μm, among which Example 10-2, Example 10-7 to Example 10-9>4μm is worse than other examples. The effective site deposition rate is 44.6% to 64.3%, with a large difference, among which Example 10-2, Example 10-3, Example 10-7 to Example 10-9 is slightly worse than other examples.

[0198] The results of the properties of the preparations encapsulating different mRNAs in Example 1, Example 10-1, and Examples 10-4 to 10-6 show that different lipid compositions encapsulating different mRNAs all have good preparation properties.

[0199] (8) Comparison of Example 1, Comparative Example 1 and Comparative Example 2 shows that the particle size of the preparation in Example 1 is the smallest, and the distribution of the nucleic acid-lipid nanoparticles is more uniform; the order of encapsulation efficiency and integrity is Example 1>Comparative Example 1>Comparative Example 2. Comparative Example 2 is a nebulized inhalation preparation, and a sieve plate nebulizer is required to atomize the solution. The shear force generated has a destructive effect on the nanoparticles, causing a significant decrease in the mRNA integrity and encapsulation efficiency; the nucleic acid-lipid nanoparticles prepared by spray drying in Comparative Example 1 have a larger mass median aerodynamic particle size, resulting in a low deposition rate in the effective site.

[0200] summary:

[0201] (1) Whether the nucleic acid-lipid nanoparticle freeze-dried powder is pre-crushed or not, the formulation characteristics of the dry powder inhaler are not much different. The particle size is 115.2nm-123.5nm, the encapsulation efficiency is 92.2%-90.1%, the integrity is 83.1%-81.3%, the mass median aerodynamic particle size is 2.53μm-2.76μm, and the effective site deposition rate is 64.3%-60.6%, all of which perform well.

[0202] (2) pH 7.0-8.0 Tris-HCl / Tris and pH 7.6 DPBS had good effects, with small particle sizes of 115.2nm-123.5nm and uniform distribution, high encapsulation efficiency of 90.1%-92.2%, high integrity of 81.3%-83.1%, mass median aerodynamic particle size of 2.53μm-3.65μm, and effective site deposition rate of 64.3%-52.4%. pH 7.6 phosphate buffer solution had a slightly worse effect, with a particle size of 129.6nm, an encapsulation efficiency of 86.8%, an integrity of 78.4%, a mass median aerodynamic particle size of 4.17μm, and an effective site deposition rate of 48.2%.

[0203] (3) Sucrose, trehalose and maltose can all be used as excipients, among which sucrose and trehalose have better effects. Specifically, after reconstitution with sucrose and trehalose, the particle size of lipid nanoparticles was 115.2nm~127.3nm, PDI was <0.1, the particle size distribution was uniform, the encapsulation rate was 91.2%~92.2%, the integrity was 82.1%~83.1%, the aerodynamic mass median was 2.53μm~2.85μm, all <5μm, and the effective site deposition rate was 55.9%~64.3%; the particle size of maltose was 154.6nm, the encapsulation rate was 82.4%, the integrity was 80.7%, the mass median aerodynamic particle size was 3.97μm, and the effective site deposition rate was 50.3%.

[0204] (4) Mannitol, lactose, chitosan, xylitol and hyaluronic acid can all be used as carriers to deliver nucleic acid lipid nanoparticles. The formulation properties are good, and mannitol and lactose have better effects in terms of mass median aerodynamic particle size and effective site deposition rate. Specifically, after reconstitution, the particle size of lipid nanoparticles is 115.2nm-127.4nm, PDI is <0.1 and the distribution is uniform, the encapsulation rate is 90.4%-92.2%, the integrity is 80.9%-83.1%, the aerodynamic mass median is 2.53μm-4.63μm and is <5μm, and the effective site deposition rate is 48.7%-64.3%. The carriers are mannitol and lactose, with aerodynamic mass medians of 2.53μm and 2.67μm, respectively, and effective site deposition rates of 64.3% and 59.4%, respectively, which are more effective.

[0205] (5) The mass ratio of lyophilized powder to carrier of 1:3, 1:2, and 1:4 can all be used to deliver nucleic acid lipid nanoparticles, and the formulation characteristics are all good. The mass ratio of median aerodynamic particle size and effective site deposition rate is 1:3, which is more effective. Specifically, after reconstitution, the particle size of lipid nanoparticles is 115.2nm~120.4nm, PDI<0.1, encapsulation efficiency is 90.4%~92.2%, integrity is 80.3%~83.1%, the median aerodynamic mass is 2.53μm~3.76μm, all <5μm, and the effective site deposition rate is 52.2%~64.3%. Among them, the mass ratio of 1:3 has a median aerodynamic mass of 2.53μm and a deposition rate of 64.3% in the effective site, which is more effective.

[0206] (6) The addition of leucine significantly improved the formulation characteristics, mass median aerodynamic particle size and effective site deposition rate. The mass ratio of lyophilized powder to 10:0.5-10:3 was better. The addition ratio of leucine was 10:0.5-10:3, the aerodynamic mass median diameter was 2.06μm-2.37μm, the effective site deposition rate was 65.7%-67.7%, and the effect was better.

[0207] (7) Dry powder inhalers prepared using different cationic lipids SM-102, ALC-0315, D-Lin-MC3-DMA, YK-401, YK-305 and DOTAP can also be delivered, among which SM-102, YK-401, YK-305 and DOTAP as cationic lipids have better particle size distribution, encapsulation efficiency, integrity, mass median aerodynamic particle size and effective site deposition rate than ALC-0315 and D-Lin-MC3-DMA. The particle size distribution, encapsulation efficiency, integrity, mass median aerodynamic particle size and effective site deposition rate of dry powder inhalers prepared by using different neutral lipids DOPE and DPPC to replace DSPC, and using conjugated lipid ALC-0159 to replace DMG-PEG2000 are slightly worse than the lipid composition of Example 1.

[0208] (8) The dry powder inhaler prepared by the present invention has good effects and better properties than the dry powder inhaler prepared by the spray freeze drying method. The outstanding effects are small mass median aerodynamic particle size and high deposition rate in the effective part. The outstanding effects compared with the nebulized inhaler are high mRNA encapsulation rate and integrity.

[0209] Test Example 2

[0210] This test example tests the biological properties of the nucleic acid-lipid nanoparticle dry powder inhalation prepared in Example 1 and Example 10. The test index is the transfection efficiency, and the test method is as follows.

[0211] First, add 200 ng of the test sample preparation to the cell culture medium of a 24-well plate and continue to culture for 18 to 24 hours. Then collect the cell extract protein, separate the protein sample by polyacrylamide gel electrophoresis (SDS-PAGE), and transfer it to a solid phase carrier (nitrocellulose membrane). The protein on the solid phase carrier is used as an antigen to react with a specific antibody, and then react with a secondary antibody labeled with horseradish peroxidase. The substrate is developed and the protein component expressed by the specific target gene is detected by a multifunctional imager.

[0212] The nucleic acid-lipid nanoparticle dry powder inhalers prepared in Example 1 and Example 10 respectively encapsulated eGFP mRNA, mRNA with a nucleic acid sequence as shown in SEQ ID NO.1, and mRNA with a nucleic acid sequence as shown in SEQ ID NO.2. The transfection efficiency test results are shown in Table 14.

[0213] Table 14

[0214]

[0215]

[0216] Comparing Example 1 with Example 10, it can be seen that the cells can be successfully transfected and the target protein band can be detected using different lipid preparations. Among them, Example 10-2, Example 10-3, Example 10-7 to Example 10-9 are slightly worse than other examples, and the difference in encapsulating different mRNAs is small.

[0217] The dry powder inhaler prepared by using different cationic lipids SM-102, ALC-0315, D-Lin-MC3-DMA, YK-401, YK-305 and DOTAP can also be delivered. The transfection effect shows that SM-102, YK-401, YK-305 are consistent with YK-009, and ALC-0315, D-Lin-MC3-DMA, and DOTAP are slightly worse than YK-009. The dry powder inhaler was prepared by replacing DSPC with different neutral lipids DOPE and DPPC, and replacing DMG-PEG2000 with conjugated lipid ALC-0159. The transfection effect shows that the effect of replacing neutral lipids and conjugated lipids is slightly worse.

[0218] In summary, the present invention provides a nucleic acid-lipid nanoparticle dry powder inhaler, which has the following characteristics: 1) the particle size of the nucleic acid nanoparticle is 115.2nm, PDI<0.1, 2) the encapsulation rate is high>90%, 3) the integrity is more than 80%, 4) the mass median aerodynamic particle size is 2.53μm, 5) the effective site deposition rate reaches 60%, 6) the cell transfection efficiency is high. The preparation method of the inhaler provided by the present invention is simple and suitable for industrial production.

[0219] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

[0220]

[0221]

Claims

1. A nucleic acid-lipid nanoparticle dry powder inhaler, characterized in that: The inhaler comprises a nucleic acid-lipid nanoparticle lyophilized powder and a powder inhaler carrier, wherein the nucleic acid-lipid nanoparticle lyophilized powder comprises nucleic acid, lipid, buffer salt and excipient, wherein the lipid comprises a cationic lipid, a neutral lipid, an auxiliary lipid and a conjugated lipid; The neutral lipid is distearoylphosphatidylcholine, the auxiliary lipid is cholesterol, the conjugated lipid is dimyristoylglycerol-3-methoxypolyethylene glycol 2000, the molar ratio of the cationic lipid, the neutral lipid, the auxiliary lipid and the conjugated lipid is 49:10:39.5:1.5, the buffer salt is Tris-HCl / Tris, the excipient is sucrose, the powder aerosol carrier is mannitol and / or lactose, and the mass ratio of the nucleic acid-lipid nanoparticle freeze-dried powder to the powder aerosol carrier is 1:

3.

2. The inhalant according to claim 1, characterized in that The inhaler also includes leucine.

3. The inhalant according to claim 2, characterized in that The mass ratio of the nucleic acid-lipid nanoparticle freeze-dried powder to leucine is 10:(0.5-4), preferably 10:(1-3).

4. The inhalant according to any one of claims 1 to 3, characterized in that The cationic lipids include any one or a combination of at least two of the molecules having the following structures:

5. The inhalant according to any one of claims 1 to 4, characterized in that The inhalation agent is administered by oral inhalation using a quasi-inhaler; Preferably, the pH of the buffer salt is 6 to 8; Preferably, the particle size D50 of the powder aerosol carrier is 80 to 120 μm; Preferably, the mass ratio of nucleic acid to lipid is 1:(10-40); Preferably, the mass ratio of the nucleic acid to the buffer salt is 1:(15-50); Preferably, the mass ratio of the nucleic acid to the excipient is 1:(300-1000).

6. The inhalant according to any one of claims 1 to 5, characterized in that The nucleic acid includes deoxyribonucleic acid and / or ribonucleic acid; Preferably, the ribonucleic acid comprises any one or a combination of at least two of small interfering RNA, asymmetric interfering RNA, micro RNA, Dicer-substrate RNA, small hairpin RNA, messenger RNA, long non-coding RNA, circular RNA, self-amplifying RNA or 5'-pppRNA; Preferably, the nucleotides of the nucleic acid are any one of eGFP mRNA, the sequence as shown in SEQ ID NO.1, or the sequence as shown in SEQ ID NO.

2.

7. A method for preparing an inhalant according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: (1) preparing an aqueous phase containing nucleic acid and an organic phase containing lipid, and encapsulating the nucleic acid using microfluidics to obtain a nucleic acid-lipid nanoparticle solution; (2) dialyzing the nucleic acid-lipid nanoparticle solution in a buffer solution; (3) adding a freeze-drying protective agent; (4) freeze-drying the solution obtained in step (3) to obtain a freeze-dried powder; (5) The freeze-dried powder is pre-crushed or not crushed and mixed with a powder inhaler carrier to obtain a dry powder inhaler.

8. The preparation method according to claim 7, characterized in that: Step (5) further comprises the step of mixing with leucine; Preferably, the moisture content after freeze-drying in step (4) is ≤1.0%.

9. Use of the inhalant according to any one of claims 1 to 6 in the preparation of nucleic acid drugs.

10. Use of the inhalant according to any one of claims 1 to 6 in the preparation of a medicament for treating a disease or condition in a mammal; Preferably, the disease or condition comprises any one of an infectious disease, a proliferative disease, a genetic disease, an autoimmune disease, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease or a metabolic disease; Preferably, the disease is cancer; Preferably, the disease is diabetes; Preferably, the infectious disease includes any one of a disease caused by coronavirus, a disease caused by influenza virus, a disease caused by HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies or herpes.

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