Nucleic acid freeze-drying preparation and preparation method thereof

Abstract

The invention relates to a nucleic acid lyophilized preparation and a preparation method thereof, the nucleic acid lyophilized preparation comprises nucleic acid molecules, a carrier and a protective agent, the carrier is a branched histidine-lysine polymer, the protective agent is trehalose and / or sucrose, the mass ratio of the carrier to the nucleic acid molecules is (1-3.8): 1, and the mass ratio of the carrier to the protective agent is 1: (35-150). When the sucrose or the trehalose is selected as the protective agent, the mass ratio of the HKP, the nucleic acid molecules and the protective agent is optimized, so that the storage stability of the small interfering nucleic acid freeze-drying preparation is remarkably improved, the small interfering nucleic acid freeze-drying preparation can be kept at normal temperature for a long time, and the temperature requirements during storage and transportation of the small interfering nucleic acid freeze-drying preparation are reduced; the invention is of great significance to development, application and popularization of small interfering nucleic acid drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid preparations, and in particular relates to a freeze-dried nucleic acid preparation and a preparation method thereof. Background Art

[0002] The once simple concept that RNA was just a passive carrier of genetic information from DNA to protein has been revolutionized through decades of research. With the discovery of various RNA molecules and their different functions in regulating gene expression, RNA is now considered a key mediator in almost all gene expression pathways. With these great discoveries comes the huge potential of using RNA as a therapeutic approach, as it can find drugs for many targets that were once considered "undruggable" by traditional medical drugs. RNA therapy can also eliminate the risk of inserted gene mutations compared to DNA-based gene therapy.

[0003] Although RNA therapy holds great promise, one of the biggest challenges is safely delivering therapeutic RNA to the exact location of the targeted cell. RNA molecules themselves have difficulty penetrating cell membranes due to their highly negatively charged nature and relatively bulky size. In addition, they are highly unstable, and typical storage conditions require extremely low temperatures (for example, siRNA solids need to be stored at -20°C).

[0004] In order to overcome these limitations, various nanoparticle carriers are being actively developed around the world. Among them, peptide nanoparticles (PNPs) are another type of nanomaterial at the forefront of RNA drug delivery research. The advantages of peptide nanoparticles include scalability, diversity, precise adjustability, reliability, biodegradability, and good drug release.

[0005] PNP is a nanoparticle formed by the self-assembly of HKP and nucleic acid molecules. PNP plays multiple roles in small nucleic acid drugs. HKP can form nanoparticles with small interfering nucleic acids, which not only improves the stability of small interfering nucleic acids, but also helps small interfering nucleic acid drugs enter the cytoplasm of target cells and exert the effect of gene silencing. Because HKP is positively charged, it can complex with negatively charged RNA molecules mainly through electrostatic effects and wrap the RNA inside the nanoparticles. PNP preparations wrap and fix nucleic acid molecules, which can effectively improve the stability of nucleic acid molecules (the long-term storage temperature is increased from -20°C to 4°C).

[0006] PNP preparations have been used in clinical practice. For example, STP705 and STP707 of Sanno Pharmaceuticals both use PNP technology.

[0007] Although PNP preparations have made significant progress, there are still many challenges. One of them is how to further improve the stability of PNP preparations. Although the stability of typical PNP preparations has been greatly improved compared to the stability of naked nucleic acid molecules, the temperature (4°C) still has high requirements for the transportation and storage of such drugs. Summary of the invention

[0008] The purpose of the present invention is to provide a freeze-dried nucleic acid preparation with good stability in long-term storage at room temperature and a preparation method thereof.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a freeze-dried nucleic acid preparation, which includes a nucleic acid molecule, a carrier and a protective agent, wherein the carrier is a branched histidine-lysine polymer, the protective agent is trehalose and / or sucrose, the mass ratio of the carrier to the nucleic acid molecule is (1-3.8):1, and the mass ratio of the carrier to the protective agent is 1:(35-150).

[0011] Preferably, the lyophilized nucleic acid preparation consists of the nucleic acid molecule, a carrier and a protective agent.

[0012] Preferably, the mass ratio of the vector to the nucleic acid molecule is (1.5-3.5):1, for example, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1.

[0013] Preferably, the mass ratio of the carrier to the protective agent is 1:(40-120), for example 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120.

[0014] More preferably, the mass ratio of the vector to the nucleic acid molecule is (2-3):1.

[0015] Still more preferably, the mass ratio of the carrier to the protective agent is 1:(40-80).

[0016] More preferably, the mass ratio of the carrier to the protective agent is 1: (40-60). Too low a dosage of the protective agent cannot achieve the desired effect, while too high a dosage does not further improve stability and may further increase particle size and reduce the percentage of nucleic acid molecule mass.

[0017] Preferably, the nucleic acid molecule includes one or more of a messenger nucleic acid molecule (mRNA), a small interfering nucleic acid molecule (siRNA), a micronucleic acid molecule (miRNA), a small activating nucleic acid molecule (saRNA), an antisense oligonucleotide molecule (ASO) or an aptamer.

[0018] More preferably, the nucleic acid molecule is siRNA.

[0019] According to some embodiments of the present invention, the nucleic acid molecule is a combination of siRNA targeting TGF-β1 and siRNA targeting COX-2.

[0020] Further preferably, the mass ratio of the siRNA targeting TGF-β1 and the siRNA targeting COX-2 is (0.5-1.5):1, for example 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1.

[0021] Still more preferably, the mass ratio of the siRNA targeting TGF-β1 to the siRNA targeting COX-2 is (0.8-1.2):1, more preferably (0.9-1.1):1, and most preferably 1:1.

[0022] According to some embodiments of the present invention, the siRNA targeting TGF-β1 has the following nucleotide sequence:

[0023] Sense strand: 5'-CCCAAGGGCUACCAUGCCAACUUCU-3'

[0024] Antisense strand: 5′-AGAAGUUGGCAUGGUAGCCCUUGGG-3′.

[0025] According to some embodiments of the present invention, the siRNA targeting COX-2 has the following nucleotide sequence:

[0026] Sense strand: 5'-GGUCUGGUGCCUGGUCUGAUGAUGU-3'

[0027] Antisense strand: 5′-ACAUCAUCAGACCAGGCACCAGACC-3′.

[0028] Preferably, the carrier comprises HKP and / or HKP+H, and the HKP structural formula is as follows:

[0029]

[0030] K is lysine, H is histidine;

[0031] The HKP+H structural formula is as follows:

[0032]

[0033] K is lysine and H is histidine.

[0034] The particle size of the nucleic acid freeze-dried preparation of the present invention does not exceed 200nm, PDI is less than 1, and the Zeta potential is 15-35mV.

[0035] More preferably, the particle size of the lyophilized nucleic acid preparation does not exceed 180 nm, the PDI is less than 0.8, and the Zeta potential is 15 to 32 mV.

[0036] More preferably, the particle size of the lyophilized nucleic acid preparation does not exceed 150 nm, the PDI is less than 0.5, and the Zeta potential is 20 to 30 mV.

[0037] The second aspect of the present invention also provides a method for preparing the above-mentioned nucleic acid freeze-dried preparation, firstly, mixing an aqueous solution containing the nucleic acid molecule and an aqueous solution containing the carrier by a microfluidic device to form a nanoparticle solution, and then mixing the nanoparticle solution with an aqueous solution containing a protective agent, and freeze-drying to obtain the nucleic acid freeze-dried preparation.

[0038] According to some specific embodiments of the present invention, the freeze-drying procedure is as follows:

[0039] (1) Prefreezing: Prefreeze at -1°C to 1°C for 5 to 15 minutes, cool to -6°C to -4°C at a cooling rate of 0.2 to 0.4°C / min, continue to cool to -50°C to -40°C at a cooling rate of 0.6 to 1.5°C / min, and prefreeze at -50°C to -40°C for 5 to 8 hours;

[0040] (2) Drying: Dry at -50°C to -40°C for 30 to 50 min under a vacuum degree of 0.1 to 0.2 mbar, heat to -30°C to -20°C at a heating rate of 0.6 to 1.5°C / h, dry at -30°C to -20°C for 68 to 78 h, continue to heat to 20°C to 30°C at a heating rate of 45 to 55°C / h, and dry at 20°C to 30°C for 8 to 12 h.

[0041] The present invention has at least the following beneficial effects:

[0042] When sucrose or trehalose is selected as a protective agent in the present invention, the mass ratio of the branched histidine-lysine polymer, the nucleic acid molecule and the protective agent is optimized, so that the storage stability of the small interfering nucleic acid freeze-dried preparation is significantly improved, and the preparation can be kept for a long time at room temperature, thereby reducing the temperature requirements for the storage and transportation of the small interfering nucleic acid freeze-dried preparation. The present invention is of great significance to the development, application and promotion of small interfering nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the mechanism of stabilizing nanoparticles of the present invention (taking HKP and trehalose as examples). DETAILED DESCRIPTION

[0044] In order to solve the problems of poor stability of existing PNP preparations during storage and transportation at room temperature, reduced purity of nucleic acid molecules and easy inactivation, the inventors of the present application have conducted a lot of research and experimental exploration on preparation optimization. In the preliminary experiments, the inventors of the present application have tried the ratio of preparation carriers and nucleic acid molecules and the addition of protective agents, but all failed to effectively improve the stability of the preparation. For example, the inventors of the present application have tried to increase the ratio of branched histidine-lysine polymer carriers, which can improve the solution stability of PNP preparations, but the long-term storage stability in the freeze-dried state has not been improved, while reducing the ratio of branched histidine-lysine polymer carriers significantly reduces the solution stability of PNP preparations and the long-term storage stability of freeze-dried preparations. For another example, adding freeze-drying protective agents (maltodextrin, microcrystalline cellulose, trehalose, glucose, dextran, sucrose, mannitol, sorbitol, xylitol, etc.) on the basis of the existing PNP preparation formula has not been very effective in improving the room temperature storage stability of PNP preparations. By accident, the inventors of the present application found that by reducing the proportion of the branched histidine-lysine polymer carrier and adding an appropriate amount of trehalose or sucrose, the obtained preparation has unexpectedly high stability and good properties of the freeze-dried preparation, and still has good properties, high siRNA purity and activity after being stored at room temperature for up to 24 months. Through further exploration, the technical solution of the present invention is proposed, which is as follows:

[0045] A nucleic acid freeze-dried preparation comprises a nucleic acid molecule, a carrier and a protective agent, wherein the carrier is a branched histidine-lysine polymer, and the protective agent is trehalose and / or sucrose, the mass ratio of the carrier to the nucleic acid molecule is (1-3.8):1, and the mass ratio of the carrier to the protective agent is 1:(35-150).

[0046] Further preferably, the nucleic acid freeze-dried preparation consists of a nucleic acid molecule, a carrier and a protective agent, the carrier is composed of a branched histidine-lysine polymer, and more preferably, the mass ratio of the carrier to the nucleic acid molecule is (2-3):1, and more preferably, the mass ratio of the carrier to the protective agent is 1:(40-80).

[0047] After further investigation, taking HKP and trehalose as examples, the mechanism of stabilizing the nanoparticles of the present invention is as follows: Figure 1 shown.

[0048] The technical solution, implementation process and principle will be further explained below.

[0049] The technical scheme and technical effects of the present invention are further described below in conjunction with specific embodiments and comparative examples.

[0050] Unless otherwise specified, the raw materials and the like used in the following examples were commercially available.

[0051] The small interfering nucleic acid used in the following examples and comparative examples is a mixture of a specific small interfering nucleic acid targeting TGF-β1 and a specific small interfering nucleic acid targeting COX-2 at a mass ratio of 1:1.

[0052] Among them, the sequence of the specific small interfering nucleic acid targeting TGF-β1 is as follows:

[0053] Sense strand:5'-CCCAAGGGCUACCAUGCCAACUUCU-3' (SEQ ID NO: 1),

[0054] Antisense strand:5'-AGAAGUUGGCAUGGUAGCCCUUGGG-3' (SEQ ID NO: 2);

[0055] The sequence of the specific small interfering nucleic acid targeting COX-2 is as follows:

[0056] Sense strand:5'-GGUCUGGGCCCUGGUCUGAUGAUGU-3' (SEQ ID NO: 3),

[0057] Antisense strand: 5'-ACAUCAUCAGACCAGGCACCAGACC-3' (SEQ ID NO: 4).

[0058] The structural formula of HKP used in the following examples and comparative examples is as follows:

[0059]

[0060] Unless otherwise specified, the room temperature (RT) referred to in the following examples refers to 20-25°C.

[0061] Unless otherwise specified, the experimental methods or test methods involved in the following embodiments adopt conventional methods in the art.

[0062] Examples 1 to 14 and Comparative Examples 1 to 17 provide freeze-dried small interfering nucleic acid preparations of different formulations, and the components and proportions are shown in Table 1.

[0063] Table 1

[0064]

[0065]

[0066] Note: “ / ” in Table 1 means not used, and the corresponding ratio is HKP:siRNA (mass ratio).

[0067] The preparation method of the siRNA freeze-dried preparation is as follows:

[0068] Ultrapure water and siRNA were used to prepare siRNA solutions with a concentration of 1 mg / mL, ultrapure water and HKP were used to prepare HKP solutions with concentrations of 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, and 6 mg / mL, and ultrapure water and protective agents were used to prepare 50% protective agent aqueous solution (50 g / 100 mL). According to the mass ratio of HKP and siRNA in Table 1, a suitable HKP solution was selected, and it was mixed with the siRNA solution in a volume ratio of 1:1 by a microfluidic process to obtain a PNP solution. Part of the PNP solution was freeze-dried to obtain the small interfering nucleic acid freeze-dried preparations of Comparative Examples 1 to 5, and 1 mL of the other part of the PNP solution was added to a vial, and a suitable 50% protective agent aqueous solution was added to the corresponding vial according to the formulation in Table 1, and freeze-dried to obtain a small interfering nucleic acid freeze-dried preparation. The freeze-drying procedure is shown in Table 2.

[0069] Table 2

[0070]

[0071]

[0072] In Table 2, the time in steps (1), (4), (7), (9), and (11) is the time for maintaining the corresponding temperature. The time in steps (2), (3), (8), and (10) is the time used to adjust the temperature from the previous step to the required temperature of the current step. Steps (5) and (6) are performed simultaneously. The vacuum degree "-" represents normal pressure (one standard atmospheric pressure, 1.013×10 3 mbar).

[0073] Taking the siRNA freeze-dried preparation of Example 1 (HKP: siRNA: trehalose = 1.5: 1: 60) as an example, the preparation method is specifically as follows: 1 mg / mL siRNA solution and 1.5 mg / mL HKP solution are mixed by a microfluidic process, the total mixing flow rate is 14 mL / min, the flow rate ratio is 1 / 1, and HKP / siRNA = 1.5 PNP solution is obtained, and then 1 mL / vial is dispensed into vials, 0.06 mL of 50% trehalose aqueous solution is added to each vial, and the mixture is evenly mixed and freeze-dried.

[0074] At least 9 vials were prepared for each embodiment and comparative example, and performance tests were performed on the day (0 day) when the siRNA freeze-dried preparation was prepared, after 6 months of storage at room temperature, and after 12 months of storage at room temperature. Three replicates were set for each test, and the average value was taken. The test method was as follows: 1 mL of water for injection was added to the vial to reconstitute the siRNA freeze-dried preparation, and the particle size (Particle size / nm), PDI (polydispersity index) and Zeta potential (Zeta potential / mV) were detected using a ZetasizerNano ZS nano-laser particle size analyzer, and the purity of the siRNA was calculated using anion exchange chromatography and the area normalization method. The test results are summarized in Table 3.

[0075] Table 3

[0076]

[0077]

[0078]

[0079] Note: "NA" in Table 3 means not tested, because the product obviously does not meet the quality requirements and was not tested.

[0080] The basic quality requirements for the freeze-dried preparations of small interfering nucleic acids in Table 3 include: particle size not exceeding 200 nm, PDI < 1, Zeta potential 15-35 mV, maintaining powder form and basically no color change.

[0081] According to Tables 1 and 3, when sucrose or trehalose is selected as the protective agent, the mass ratio of HKP, siRNA and the protective agent is optimized, and the storage stability of the siRNA freeze-dried preparation is significantly improved, and its properties and activity can be maintained for a long time at room temperature, thereby reducing the temperature requirements for the storage and transportation of the siRNA freeze-dried preparation. The present invention is of great significance to the development, application and promotion of siRNA drugs.

Claims

1. A freeze-dried nucleic acid preparation, characterized in that: The nucleic acid freeze-dried preparation comprises a nucleic acid molecule, a carrier and a protective agent, wherein the carrier is a branched histidine-lysine polymer, the protective agent is trehalose and / or sucrose, the mass ratio of the carrier to the nucleic acid molecule is (1-3.8):1, and the mass ratio of the carrier to the protective agent is 1:(35-150).

2. The freeze-dried nucleic acid preparation according to claim 1, characterized in that: The nucleic acid freeze-dried preparation consists of the nucleic acid molecule, a carrier and a protective agent.

3. The freeze-dried nucleic acid preparation according to claim 1, characterized in that: The mass ratio of the carrier to the nucleic acid molecule is (1.5-3.5):1; and / or the mass ratio of the carrier to the protective agent is 1:(40-120).

4. The freeze-dried nucleic acid preparation according to claim 3, characterized in that: The mass ratio of the vector to the nucleic acid molecule is (2-3):1; and / or the mass ratio of the vector to the protective agent is 1:(40-80).

5. The freeze-dried nucleic acid preparation according to claim 1, characterized in that: The nucleic acid molecule includes one or more of a messenger nucleic acid molecule, a small interfering nucleic acid molecule, a micronucleic acid molecule, a small activating nucleic acid molecule, an antisense oligonucleotide molecule or an aptamer.

6. The freeze-dried nucleic acid preparation according to claim 1, characterized in that: The nucleic acid molecule is a combination of siRNA targeting TGF-β1 and siRNA targeting COX-2.

7. The freeze-dried nucleic acid preparation according to claim 6, characterized in that: The mass ratio of the siRNA targeting TGF-β1 to the siRNA targeting COX-2 is (0.5-1.5):1; And / or, the siRNA targeting TGF-β1 has the following nucleotide sequence: Sense strand: 5'-CCCAAGGGCUACCAUGCCAACUUCU-3' Antisense strand: 5′-AGAAGUUGGCAUGGUAGCCCUUGGG-3′; And / or, the siRNA targeting COX-2 has the following nucleotide sequence: Sense strand: 5'-GGUCUGGUGCCUGGUCUGAUGAUGU-3' Antisense strand: 5′-ACAUCAUCAGACCAGGCACCAGACC-3′.

8. The freeze-dried nucleic acid preparation according to claim 1, characterized in that: The carrier includes HKP and / or HKP+H, and the HKP structural formula is as follows: K is lysine, H is histidine; The HKP+H structural formula is as follows: K is lysine and H is histidine.

9. The method for preparing a lyophilized nucleic acid preparation according to any one of claims 1 to 8, characterized in that: First, an aqueous solution containing the nucleic acid molecule and an aqueous solution containing the carrier are mixed by a microfluidic device to form a nanoparticle solution, and then the nanoparticle solution is mixed with an aqueous solution containing a protective agent, and freeze-dried to obtain the nucleic acid freeze-dried preparation.

10. The method for preparing a lyophilized nucleic acid preparation according to claim 9, characterized in that: The freeze-drying procedure is as follows: (1) Prefreezing: Prefreeze at -1°C to 1°C for 5 to 15 minutes, cool down to -6°C to -4°C at a cooling rate of 0.2 to 0.4°C / min, continue to cool down to -50°C to -40°C at a cooling rate of 0.6 to 1.5°C / min, and prefreeze at -50°C to -40°C for 5 to 8 hours; (2) Drying: Dry at -50°C to -40°C for 30 to 50 min under a vacuum degree of 0.1 to 0.2 mbar, heat to -30°C to -20°C at a heating rate of 0.6 to 1.5°C / h, dry at -30°C to -20°C for 68 to 78 h, continue to heat to 20°C to 30°C at a heating rate of 45 to 55°C / h, and dry at 20°C to 30°C for 8 to 12 h.

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