A nucleic acid-lipid nanoparticle dry powder inhaler, and methods of making and using the same

Abstract

This invention relates to a nucleic acid-lipid nanoparticle dry powder inhaler, its preparation method, and its application. The inhaler comprises a lyophilized nucleic acid-lipid nanoparticle powder and a powder carrier. The lyophilized nucleic acid-lipid nanoparticle powder includes nucleic acids, lipids, buffer salts, and excipients. The lipids include cationic lipids, neutral lipids, auxiliary lipids, and conjugated lipids. The nucleic acid nanoparticles in the inhaler provided by this invention have a particle size controlled at 115 nm, an encapsulation efficiency of over 90%, nucleic acid integrity of over 80%, a median aerodynamic particle size of 2.53 μm, an effective site deposition rate of over 60%, high cell transfection efficiency, and a simple preparation method suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparations, and more particularly to a nucleic acid-lipid nanoparticle dry powder inhaler, its preparation method, and its application. Background Technology

[0002] The lungs are the most vulnerable internal organs to infection and damage from the external environment, frequently exposed to particles, chemicals, and infectious microorganisms in the ambient air. Respiratory diseases impose a huge health burden worldwide, and since the COVID-19 pandemic, the number of cases of pulmonary dysfunction has increased on top of all other lung diseases, inevitably leading to a greater need for novel pulmonary therapies that are applied topically to the site of action.

[0003] RNA therapy holds promise for treating respiratory diseases, offering the potential for highly selective, potent, and personalized treatment compared to traditional therapies using proteins, peptides, small molecules, or monoclonal antibodies. RNA is a negatively charged macromolecule that does not bind to the cell surface or cross the cell membrane. However, RNA therapy also faces challenges such as nuclease degradation, off-target gene silencing, and immunostimulatory effects.

[0004] To overcome these limitations, RNA is encapsulated in a variety of materials, including lipids, polymers, inorganic materials, proteins, and combinations thereof. Lipid-based powder aerosol carrier systems mimicking pulmonary surfactant or cell membranes enhance the ability to overcome the lung's biological barrier and reduce toxicity and antigenicity. While all commercially available RNA drugs target the liver, the lungs offer a variety of currently untreatable targets that could potentially be treated with RNA therapy. Therefore, local pulmonary delivery of RNA nanoparticles could ultimately achieve delivery outside the liver. Administration of RNA drugs via dry powder inhalers offers numerous advantages related to the physical, chemical, and microbiological stability of RNA and nanosuspensions. Furthermore, local delivery to the lungs offers significant advantages over systemic administration, reducing the dosage required for local effects and thus minimizing side effects.

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

[0006] In summary, developing a high-quality nucleic acid-loaded formulation and applying it to lung administration to address the high incidence of lung diseases and provide more options for clinical medication has become one of the urgent problems to be solved in this field. Summary of the Invention

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

[0008] To achieve this objective, the present invention adopts the following technical solution:

[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, wherein the nucleic acid-lipid nanoparticle lyophilized powder comprises nucleic acid, lipid, buffer salt and excipient, and the lipid comprises cationic lipid, neutral lipid, auxiliary lipid and conjugated lipid;

[0010] The neutral lipid is distearate phosphatidylcholine (DSPC), the auxiliary lipid is cholesterol, the conjugated lipid is dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (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 atomizer carrier is mannitol and / or lactose, and the mass ratio of the nucleic acid-lipid nanoparticle lyophilized powder to the powder atomizer carrier is 1:3.

[0011] Preferably, the inhalant also includes leucine.

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

[0013] The specific point values ​​for (0.5 to 4) can be 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4, etc.

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

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

[0016]

[0017]

[0018] Preferably, the inhalant is administered orally via a nasal inhaler.

[0019] Preferably, the pH of the buffer salt is 6 to 8 (e.g., it can be 6, 6.5, 7, 7.5 or 8, etc.), and more preferably the pH is 7.6.

[0020] Preferably, the particle size D50 of the powder atomizer carrier is 80-120 μm (e.g., it can 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 ​​for (10-40) can be selected as 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 ​​for (15-50) can be selected as 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 ​​for 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 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 include 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, and 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. The mass ratio of the nucleic acid-lipid nanoparticle lyophilized powder to the powder inhaler carrier is 1:3.

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

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

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

[0031] In a second aspect, the present invention provides a method for preparing an inhalant as described in the first aspect, the method comprising the following steps:

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

[0033] (2) Dialysis of nucleic acid-lipid nanoparticle solution in buffer solution;

[0034] (3) Add freeze-drying protectant;

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

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

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

[0038] (a) Preparation of aqueous phase:

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

[0040] (b) Preparation of organic phase:

[0041] The organic phase was prepared by dissolving cationic lipids, neutral lipids, auxiliary lipids and conjugated lipids in anhydrous ethanol at a molar ratio of 49:10:39.5:1.5.

[0042] (c) Encapsulation:

[0043] The organic and aqueous phases were rapidly mixed and encapsulated in a microfluidic device. The encapsulation method involved an organic phase to aqueous phase inlet flow rate ratio of 1:(2-4) and a total inlet flow rate of 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 solution to obtain mRNA lipid nanoparticle (mRNA-LNP) samples.

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

[0047] The excipient was dissolved in nuclease-free water to prepare a freeze-drying protectant solution.

[0048] (f) Preparation of mRNA-LNP lyophilized formulations using freeze-drying protectants

[0049] The freeze-drying protectant solution prepared in step (e) is added to the mRNA-LNP sample in step (d) to obtain a freeze-drying mixture with an excipient concentration of 5-15%. The solution is then freeze-dried to control the moisture content to <1.0% to obtain a nucleic acid-lipid nanoparticle freeze-dried powder.

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

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

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

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

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

[0055] Fourthly, the present invention provides the use of the inhalant as described in the first aspect in the preparation of a medicament for treating diseases or conditions in mammals.

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

[0057] Preferably, the disease is cancer.

[0058] Preferably, the disease is diabetes.

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

[0060] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

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

[0062] This invention provides a nucleic acid-lipid nanoparticle dry powder inhaler, comprising a nucleic acid-lipid nanoparticle lyophilized powder and a powder carrier. The nucleic acid-lipid nanoparticle lyophilized powder comprises nucleic acid, lipids, a buffer salt, and an excipient. The lipids include YK-009, DSPC, cholesterol, and DMG-PEG2000, with a molar ratio of 49:10:39.5:1.5. The buffer salt is Tris-HCl / Tris, the excipient is sucrose, and the powder carrier is mannitol. The mass ratio of the nucleic acid-lipid nanoparticle lyophilized powder to the powder carrier is 1:3. This invention achieves significant technical effects by using specific excipients, specific carriers, and specific nucleic acid lipid nanoparticles to form a whole, thereby improving the deposition rate and encapsulation efficiency of effective sites. It has the following characteristics: 1) Nucleic acid nanoparticles with a particle size of 115.2 nm and a PDI < 0.1; 2) High encapsulation efficiency > 90%; 3) Integrity > 80%; 4) Median aerodynamic particle size of 2.53 μm; 5) Effective site deposition rate reaches 60%; 6) Significantly improved cell transfection efficiency. Specifically:

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

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

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

[0066] (4) Carrier Types: Mannitol, lactose, chitosan, xylitol, and hyaluronic acid can all be used as carriers for delivering nucleic acid lipid nanoparticles, all exhibiting good formulation characteristics. Mannitol and lactose show superior performance in terms of median aerodynamic particle size and effective site deposition rate. Specifically, after reconstitution, the lipid nanoparticles have a particle size of 115.2 nm–127.4 nm, a PDI of <0.1, uniform distribution, encapsulation efficiency of 90.4%–92.2%, integrity of 80.9%–83.1%, a median aerodynamic mass of 2.53 μm–4.63 μm (all <5 μm), and an effective site deposition rate of 48.7%–64.3%. Mannitol, as the carrier, has a median aerodynamic mass of 2.53 μm and an effective site deposition rate of 64.3%, demonstrating superior performance.

[0067] (5) Carrier dosage: A 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, with good formulation characteristics. A mass ratio of 1:3 yields superior results in terms of median aerodynamic particle size and effective site deposition rate. Specifically, after reconstitution, the lipid nanoparticles have a particle size of 115.2 nm–120.4 nm, a PDI < 0.1, an encapsulation efficiency of 90.4%–92.2%, an integrity of 80.3%–83.1%, a median aerodynamic particle size of 2.53 μm–3.76 μm (all < 5 μm), and an effective site deposition rate of 52.2%–64.3%. The 1:3 mass ratio shows a median aerodynamic particle size of 2.53 μm and an effective site deposition rate of 64.3%, demonstrating superior performance.

[0068] (6) Leucine dosage: The addition of leucine significantly improves the formulation characteristics, median aerodynamic particle size, and effective fraction deposition rate. A mass ratio of lyophilized powder to leucine of 10:0.5–10:3 yields even better results. A leucine addition ratio of 10:0.5–10:3 results in a median aerodynamic particle size of 2.06 μm–2.37 μm and an effective fraction deposition rate of 65.7%–67.7%, demonstrating even better performance.

[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 are superior to ALC-0315 and D-Lin-MC3-DMA in terms of particle size distribution, encapsulation efficiency, integrity, median aerodynamic particle size and effective site deposition rate as cationic lipids. 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, 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 by replacing DMG-PEG2000 with conjugated lipid ALC-0159, were slightly worse. The transfection effect showed that both replacing neutral lipids and conjugated lipids were slightly worse. Detailed Implementation

[0070] To further illustrate the technical means and effects of this invention, the following embodiments are provided for further explanation. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0071] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0072] Definitions:

[0073] Effective site deposition rate: The effective site is determined by sieving the powder after inhalation of the formulation in an in vitro simulated inhalation drug delivery system, and statistically analyzing the content of the active ingredient under different pore sizes. Among them, particles with a diameter of less than 4.7 μm are particles that can be delivered to the lungs. Therefore, the effective site deposition rate refers to the percentage of drug contained in particles with a diameter of less than 4.7 μm relative to the total delivered dose by mass.

[0074] DSPC: Distearylphosphatidylcholine;

[0075] DMG-PEG2000: Dimyristic glycerol-3-methoxy polyethylene glycol 2000;

[0076] The experimental data in this application were analyzed using the t-test procedure in EXCEL software. P-values ​​were calculated, and P < 0.05 was considered statistically significant.

[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) Aqueous 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 organic phase was prepared by dissolving cationic lipids, neutral lipids, auxiliary lipids and conjugated lipids in anhydrous ethanol.

[0083] (3) Encapsulation:

[0084] The organic and aqueous phases were rapidly mixed and encapsulated in a microfluidic device (manufacturer: Precision NanoSystems). The encapsulation method involved an organic-to-aqueous phase influent ratio of 1:3, a total influent flow rate of 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 25mM pH 7.6 buffer to obtain mRNA lipid nanoparticle (mRNA-LNP) samples.

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

[0088] Sucrose was dissolved in nuclease-free water to prepare a freeze-drying protectant solution.

[0089] (6) Preparation of mRNA-LNP lyophilized formulations using freeze-drying protectants

[0090] The freeze-drying protectant solution prepared in step (5) is added to the mRNA-LNP sample in step (4) to obtain a freeze-drying mixture. The solution is then freeze-dried to control the moisture content to <1.0% to obtain a nucleic acid-lipid nanoparticle freeze-dried powder.

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

[0092] The above-mentioned lyophilized nucleic acid-lipid nanoparticle powder was mixed with mannitol (Pearlitol 100SD, D50: 113μm, purchased from Merck, Germany) and stirred at 150 rpm with a shear rate of 2000 rpm. After mixing was completed, the 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 according to CN114044741A), DSPC (purchased from Nippon Seika Co., Ltd.), cholesterol (purchased from Nippon Seika Co., Ltd.), and DMG-PEG2000 (purchased from Xiamen Sainobang 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 according 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 to lactose is 1:3.

[0100] Table 2

[0101]

[0102] Example 3

[0103] 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 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 embodiment prepares a nucleic acid-lipid nanoparticle dry powder inhaler. The only difference from Example 1 is that when using the preparation method provided in Preparation Example 1, the nucleic acid-lipid nanoparticle lyophilized powder obtained in step (6) is pulverized using an airflow mill, and the D90 is controlled to be 1.0-5.0 μm to obtain nucleic acid-lipid nanoparticle lyophilized powder. The lyophilized powder is mixed with mannitol in step (7) at a mass ratio of 1:3, and the mixing parameters are the same as in Preparation Example 1 to obtain the nucleic acid-lipid nanoparticle dry powder inhaler.

[0109] Example 5

[0110] This embodiment prepared a nucleic acid-lipid nanoparticle dry powder inhaler, which differed from Example 1 only in that the pH 7.6 Tris-HCl / Tris buffer was replaced with the buffer in Table 4.

[0111] Table 4

[0112] Group Types of buffer solutions 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 embodiment prepares a nucleic acid-lipid nanoparticle dry powder inhaler, which differs from Example 1 only in that the excipient sucrose is replaced with the excipients in Table 5.

[0115] Table 5

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

[0117] Example 7

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

[0119] Table 6

[0120] Group Types 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 Aiweituo

[0121] Example 8

[0122] This embodiment prepares a nucleic acid-lipid nanoparticle dry powder inhaler. The only difference from Example 1 is that the mass ratio of nucleic acid-lipid nanoparticle freeze-dried powder and mannitol is replaced by the mass ratio in Table 7 instead of 1:3.

[0123] Table 7

[0124] Group mass ratio Example 8-1 1:2 Example 8-2 1:4

[0125] Example 9

[0126] This embodiment prepares a nucleic acid-lipid nanoparticle dry powder inhaler. The only difference from Example 1 is that when using the preparation method provided in Example 1, step (7) involves mixing the nucleic acid-lipid nanoparticle freeze-dried powder, mannitol, and leucine, stirring at 150 rpm, and shearing at 2000 rpm. After mixing, the nucleic acid-lipid nanoparticle dry powder inhaler is obtained. The mass ratio of the nucleic acid-lipid nanoparticle freeze-dried powder to mannitol is 1:3. The mass ratios of the nucleic acid-lipid nanoparticle freeze-dried powder to leucine are 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 embodiment prepares a nucleic acid-lipid nanoparticle dry powder inhaler, which differs 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] This comparative example prepared a nucleic acid-lipid nanoparticle dry powder inhaler. The only difference from Example 1 is that when using the preparation method provided in Example 1, 1g of mannitol was dissolved in 46mL of water to obtain an aqueous solution of excipients. After being fully dissolved with the mRNA-LNP obtained in step (4), the solution was spray-dried to prepare lyophilized powder. The lyophilized powder and mannitol were mixed at a mass ratio of 1:3.

[0136] Comparative Example 2

[0137] This comparative example prepared a nucleic acid-lipid nanoparticle dry powder inhaler. The only difference from Example 1 is that when using the preparation method provided in Example 1, the mRNA-LNP obtained in step (4) was mixed with 0.5% poloxamer 188 and nebulized using a vibrating mesh nebulizer.

[0138] Test Example 1

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

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

[0141] Particle size and PDI were measured using dynamic light scattering (DLS) with a nano-laser particle size analyzer (manufacturer: MALVERN; model: ZSU3305) at 25±1℃. A 60μL sample was placed in a quartz micro sample cell for analysis. The analysis parameters 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 (partial dispersion index) are important characteristics for characterizing liposomes. During freeze-thaw cycles, liposomes fuse, with smaller liposomes fusing into slightly larger ones. The particle size increases with the number of freeze-thaw cycles. Within a certain range, smaller particle size results in less change in particle size after freeze-drying, indicating better product quality and processing. The dispersion index reflects the particle size distribution; a smaller dispersion index indicates more uniform liposome particle size.

[0145] (2) Encapsulation ratio:

[0146] The sample was diluted to 2.8 μg / mL. A portion was mixed with an equal volume (50 μL) of Triton X-100 and thoroughly demulsified for determining the total mRNA concentration. The other portion was mixed with an equal volume (50 μL) of TE (Tris-EDTA) for determining the concentration of free, unencapsulated mRNA. The samples with added TE and Triton X-100 were incubated at 37°C for a certain time. Then, 100 μL of Ribogreen reagent diluted 200-fold was added. After centrifugation to remove air bubbles, fluorescence was measured using a multi-plate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The plate was then read.

[0147] (3) Integrity:

[0148] mRNA was extracted from mRNA-LNP using a kit, diluted, and denatured at 65°C. After sample preparation, the sample was placed on ice. Separately, gel buffer was prepared, and the prepared sample solution, gel buffer, and ribonuclease-free water were added sequentially to the sample dish. The integrity spectrum of the sample mRNA was determined using PA800 Plus gel electrophoresis.

[0149] (4) Median aerodynamic particle size and effective deposition rate:

[0150] The Andersen disc impactor (ACI) was used to detect drug distribution and deposition during drug administration at an airflow velocity of 30 L / min. The deposition rate at effective sites (active components contained in particles smaller than 4.7 μm) and the median aerodynamic mass 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 one dose of dry powder inhaler (labeled dose is 100μg) and place it in the mouthpiece adapter.

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

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

[0155] ⑤ Use water to extract the drugs from the inner walls of each level and the corresponding collection plates.

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

[0157] Based on the amount of drug in the above solution, a cumulative deposition rate versus particle size curve was plotted.

[0158] From the above curve, we can obtain:

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

[0160] (II) Effective site deposition rate: The percentage of drug with a particle size of less than 4.7 μm by mass of the total delivered dose.

[0161] The nucleic acid-lipid nanoparticles provided in Examples 1-10 and Comparative Examples 1-2 were used to encapsulate eGFP mRNA (purchased from Genscript Biotech), and 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 mRNA shown in SEQ ID NO.1 was encapsulated using the nucleic acid-lipid nanoparticles provided in Examples 1 and 10, and the test results are shown in Table 12.

[0167] Table 12

[0168]

[0169] The mRNA shown in SEQ ID NO.2 was encapsulated using the nucleic acid-lipid nanoparticles provided in Examples 1 and 10, and the test results are shown in Table 13.

[0170] Table 13

[0171]

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

[0173] (1) Comparing Example 1 and Example 4, it can be seen that the particle size distribution of the lyophilized nucleic acid-lipid nanoparticles is relatively small whether or not they are pre-crushed; the encapsulation rate of the lyophilized nucleic acid-lipid nanoparticles is slightly lower than that of the un-crushed ones, but the decrease is small and the encapsulation rate is still >90%; the integrity of the lyophilized nucleic acid-lipid nanoparticles is slightly lower than that of the un-crushed ones, but the decrease is small and the integrity is still >80%; whether or not the lyophilized nucleic acid-lipid nanoparticles are pre-crushed, the median aerodynamic particle size is <5μm in the range of 2.53μm to 2.76μm, and the effective part deposition rate is 60.6% to 64.3%, with little difference in delivery effect.

[0174] (2) A comparison of Examples 1 and 5 shows that the dry powder inhalers prepared using different types of buffer solutions and Tris-HCl / Tris buffer solutions with different pH values ​​all had lipid nanoparticle sizes <130 nm and PDI <0.1 after reconstitution, exhibiting uniform particle size distribution. In Example 1, the smallest particle size (115.2 nm) was obtained by dialysis with pH 7.6 Tris-HCl / Tris buffer solution, followed by pH 7.6 DPBS buffer solution, while the largest particle size was obtained with pH 7.6 phosphate buffer solution. Dialysis with different pH values ​​of Tris-HCl / Tris buffer solution showed minimal particle size variation within the pH range of 7.0–8.0.

[0175] Dry powder inhalers were prepared using different types of buffer solutions. The encapsulation efficiency was >90% with pH 7.6 Tris-HCl / Tris buffer and pH 7.6 DPBS buffer, which showed good encapsulation effect on mRNA. The encapsulation efficiency was relatively low with pH 7.6 phosphate buffer. Dialysis with pH 7.0–8.0 Tris-HCl / Tris buffer showed high encapsulation efficiency.

[0176] Dialysis with pH 7.0–8.0 Tris-HCl / Tris buffer and pH 7.6 DPBS buffer showed >80% integrity, while dialysis with pH 7.6 phosphate buffer showed slightly lower integrity.

[0177] Median aerodynamic particle size analysis showed that pH 7.6 phosphate buffer was slightly less effective than pH 7.6 Tris-HCl / Tris buffer and pH 7.6 DPBS buffer. The effective deposition rate was best with pH 7.0–8.0 Tris-HCl / Tris buffer, followed by pH 7.6 DPBS buffer, and the worst with pH 7.6 phosphate buffer.

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

[0179] Using sucrose and trehalose as excipients, the encapsulation efficiency was >90%, while the encapsulation efficiency using maltose was slightly lower at 82.4%. This indicates that all three excipients showed good encapsulation effects on mRNA, with sucrose and mannitol showing better results.

[0180] When sucrose, trehalose, and maltose were used as excipients, the differences in mRNA integrity were small, and all were high, indicating that the mRNA integrity was good when the excipients were selected from the above three.

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

[0182] (4) Comparing Example 1 and Example 7, it can be seen that when mannitol, lactose, chitosan, xylitol and hyaluronic acid are used as carriers respectively, the particle size of the lipid nanoparticles after reconstitution is all <130nm, ranging from 115.2nm to 127.4nm, and the PDI is all <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, and the encapsulation rates ranged from 90.4% to 92.2%, all greater than 90%, indicating that the five carriers selected all had good encapsulation effects on mRNA.

[0184] Using mannitol, lactose, chitosan, xylitol, and hyaluronic acid as vectors, the mRNA integrity ranged from 80.9% to 83.1%, with little difference and all being relatively high, indicating that the vectors selected from the above five types had good mRNA integrity.

[0185] The median aerodynamic mass ranged from 2.53 μm to 4.63 μm, all less than 5 μm, and the effective deposition rate ranged from 48.7% to 64.3%, indicating that all five carriers selected could be used for delivery. Among them, Mannitol and Lactose were used as carriers in Examples 1 and 7-1, with median aerodynamic mass of 2.53 μm and 2.67 μm, respectively, and effective deposition rates of 64.3% and 59.4%, respectively, demonstrating superior performance.

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

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

[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 integrity of mRNA showed little difference, 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 ranged from 2.53 μm to 3.76 μm, all less than 5 μm, and the effective deposition rate ranged from 52.2% to 60.3%, indicating that a mass ratio of lyophilized powder to carrier of 1:2 to 1:4 can be used for delivery. Among them, a mass ratio of 1:3 resulted in a median aerodynamic mass of 2.53 μm and an effective deposition rate of 64.3%, demonstrating superior performance.

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

[0191] The encapsulation rates were between 93.0% and 93.3%, with little difference and all being relatively high, indicating that the encapsulation rates were good within the range of 10:0.5 to 10:4.

[0192] The integrity was between 84.0% and 84.4%, with little difference and all being relatively high, indicating that the integrity was good within the range of 10:0.5 to 10:4.

[0193] The median aerodynamic mass diameter was less than 5 μm in the range of 2.06 μm to 2.53 μm, and the effective deposition rate was 64.2% to 67.7%. This indicates that the delivery can be carried out in a mass ratio of lyophilized powder to leucine of 10:0.5 to 10:4. When the addition ratio was 10:0.5 to 10:3, the median aerodynamic mass diameter tended to decrease, while the effective deposition rate tended to increase significantly.

[0194] (7) Comparing Example 1 and Example 10, it can be seen that using different lipid compositions, Examples 10-1 to 10-6 used different cationic lipids to replace YK-009, and the particle size of the lipid nanoparticles after reconstitution showed certain differences. Among them, the cationic lipids ALC-0315 and D-Lin-MC3-DMA had significantly larger particle sizes than those in other examples, followed by DOTAP. SM-102, YK-401, and YK-305 were similar to YK-009 in Example 1. Examples 10-7 and 10-8 used different neutral lipids to replace DSPC, and Example 10-9 used conjugated lipid ALC-0159 to replace DMG-PEG2000, resulting in a poorer particle size effect than in Example 1.

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

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

[0197] Using different lipid compositions, the median aerodynamic particle size ranged from 2.53 μm to 4.88 μm, all <5 μm. Examples 10-2, 10-7, and 10-9, with particle sizes >4 μm, were inferior to the other examples. The effective deposition rate varied considerably, ranging from 44.6% to 64.3%, with Examples 10-2, 10-3, 10-7, and 10-9 showing slightly lower rates than the other examples.

[0198] Examples 1, 10-1, and 10-4 to 10-6 show that different lipid compositions encapsulating different mRNAs all exhibit good formulation properties.

[0199] (8) Comparing Example 1, Comparative Example 1 and Comparative Example 2, it can be seen that the formulation of Example 1 has the smallest particle size and the distribution of 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 formulation, which requires the use of a sieve plate nebulizer to atomize the solution. The shear force generated has a destructive effect on the nanoparticles, which significantly reduces the integrity and encapsulation efficiency of mRNA. The nucleic acid-lipid nanoparticles prepared by spray drying in Comparative Example 1 have a larger median aerodynamic particle size, resulting in a lower deposition rate of its effective site.

[0200] summary:

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

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

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

[0204] (4) Mannitol, lactose, chitosan, xylitol, and hyaluronic acid can all be used as carriers for delivering nucleic acid lipid nanoparticles, all exhibiting good formulation characteristics. Mannitol and lactose show superior performance in terms of median aerodynamic particle size and effective site deposition rate. Specifically, after reconstitution, the lipid nanoparticles have a particle size of 115.2 nm–127.4 nm, a PDI of <0.1, uniform distribution, encapsulation efficiency of 90.4%–92.2%, integrity of 80.9%–83.1%, a median aerodynamic mass of 2.53 μm–4.63 μm (all <5 μm), and an effective site deposition rate of 48.7%–64.3%. Mannitol and lactose, as carriers, have median aerodynamic mass of 2.53 μm and 2.67 μm, respectively, and effective site deposition rates of 64.3% and 59.4%, respectively, showing superior performance.

[0205] (5) Lyophilized powder to carrier ratios of 1:3, 1:2, and 1:4 can all be used to deliver nucleic acid lipid nanoparticles, with good formulation characteristics. A ratio of 1:3 shows superior performance in terms of median aerodynamic particle size and effective site deposition rate. Specifically, after reconstitution, the lipid nanoparticles have a particle size of 115.2 nm–120.4 nm, a PDI < 0.1, an encapsulation efficiency of 90.4%–92.2%, an integrity of 80.3%–83.1%, a median aerodynamic particle size of 2.53 μm–3.76 μm (all < 5 μm), and an effective site deposition rate of 52.2%–64.3%. The 1:3 ratio shows a median aerodynamic particle size of 2.53 μm and an effective site deposition rate of 64.3%, demonstrating superior performance.

[0206] (6) The addition of leucine significantly improves the formulation characteristics, median aerodynamic particle size, and deposition rate of effective fractions. The effect is better when the mass ratio of lyophilized powder to leucine is 10:0.5 to 10:3. When the leucine addition ratio is 10:0.5 to 10:3, the median aerodynamic particle size is 2.06 μm to 2.37 μm, and the deposition rate of effective fractions is 65.7% to 67.7%, with even better results.

[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 them, SM-102, YK-401, YK-305, and DOTAP, as cationic lipids, have better particle size distribution, encapsulation efficiency, integrity, median aerodynamic particle size, and effective site deposition rate than ALC-0315 and D-Lin-MC3-DMA. The particle size distribution, encapsulation efficiency, integrity, 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 by replacing DMG-PEG2000 with conjugated lipid ALC-0159, are slightly worse than the lipid composition in Example 1.

[0208] (8) The dry powder inhaler prepared by the present invention has good effects and better characteristics than the dry powder inhaler prepared by spray freeze drying method. Its outstanding effects are small median aerodynamic particle size and high deposition rate of effective site; compared with nebulized inhaler, its outstanding effects are high mRNA encapsulation rate and integrity.

[0209] Test Example 2

[0210] This test example examines the biological characteristics of the nucleic acid-lipid nanoparticle dry powder inhalers prepared in Examples 1 and 10. The test index is transfection efficiency, and the test method is as follows.

[0211] First, 200 ng of the test sample formulation was added to the cell culture medium of a 24-well plate at a rate of 24 μg per well, and the plates were incubated for 18–24 hours. Cells were then collected to extract proteins. Protein samples separated by SDS-PAGE were transferred to a solid support (nitrocellulose membrane). The proteins on the solid support were used as antigens to induce an immunoreaction with specific antibodies, followed by a reaction with horseradish peroxidase-labeled secondary antibody. The substrate was then visualized using a multi-functional imaging system to detect the protein components expressing the specific target gene.

[0212] The nucleic acid-lipid nanoparticle dry powder inhalers prepared in Examples 1 and 10 were respectively encapsulated with eGFP mRNA, mRNA with nucleic acid sequence as shown in SEQ ID NO.1, and mRNA with 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 target protein band can be detected in cells using different lipid preparations. Examples 10-2, 10-3, 10-7 to 10-9 are slightly worse than other examples, and the differences between different mRNAs encapsulated are small.

[0217] 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. Transfection efficiency showed that SM-102, YK-401, and YK-305 were consistent with YK-009, while ALC-0315, D-Lin-MC3-DMA, and DOTAP were slightly less effective than YK-009. Dry powder inhalers prepared by replacing DSPC with different neutral lipids DOPE and DPPC, and by replacing DMG-PEG2000 with the conjugated lipid ALC-0159, showed that both replacing neutral lipids and using conjugated lipids resulted in slightly poorer transfection efficiency.

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

[0219] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

[0220]

[0221]

Claims

1. A nucleic acid-lipid nanoparticle dry powder inhaler, characterized in that, The inhalant comprises a nucleic acid-lipid nanoparticle lyophilized powder and a powder inhaler carrier. The nucleic acid-lipid nanoparticle lyophilized powder comprises nucleic acids, lipids, buffer salts, and excipients. The lipids include cationic lipids, neutral lipids, auxiliary lipids, and conjugated lipids. The neutral lipid is distearylphosphatidylcholine, the auxiliary lipid is cholesterol, the conjugated lipid is dimyristoylglycerol-3-methoxy polyethylene glycol 2000, 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 with pH 7.6, the excipient is sucrose, the powder atomizer carrier is mannitol, and the mass ratio of the nucleic acid-lipid nanoparticle lyophilized powder to the powder atomizer carrier is 1:

3. The cationic lipid has the following structure: I； The inhalant is administered orally via a transinhaler. The preparation method of the inhalant includes 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 excipients; (4) freeze-drying the solution obtained in step (3) to obtain a lyophilized powder; (5) pre-crushing or not crushing the lyophilized powder and mixing it with a powder inhaler carrier to obtain a dry powder inhaler.

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).

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

5. The inhalant according to claim 1, characterized in that, The particle size D50 of the powder atomizer carrier is 80~120 μm.

6. The inhalant according to claim 1, characterized in that, The mass ratio of nucleic acid to lipid is 1:(10~40).

7. The inhalant according to claim 1, characterized in that, The mass ratio of the nucleic acid to the buffer salt is 1:(15~50).

8. The inhalant according to claim 1, characterized in that, The mass ratio of nucleic acid to excipient is 1:(300~1000).

9. The inhalant according to claim 1, characterized in that, The nucleic acids include deoxyribonucleic acid and / or ribonucleic acid.

10. The inhalant according to claim 9, characterized in that, The ribonucleic acid includes any one or a combination of at least two of the following: small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, messenger RNA, long non-coding RNA, circular RNA, self-amplifying RNA, or 5'-ppp RNA.

11. The inhalant according to claim 1, characterized in that, The nucleotide of the nucleic acid is any one of eGFP mRNA, a sequence as shown in SEQ ID NO.1, or a sequence as shown in SEQ ID NO.

2.

12. The inhalant according to claim 1, characterized in that, Step (5) also includes the step of mixing with leucine.

13. The inhalant according to claim 1, characterized in that, After freeze-drying in step (4), the moisture content is ≤1.0%.

14. The use of the inhalant as described in any one of claims 1 to 13 in the preparation of nucleic acid drugs.

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