Lyophilized nucleic acid formulation and method for preparing same
By using branched histidine-lysine polymers and trehalose or sucrose as protective agents, the formulation of nucleic acid freeze-dried preparations was optimized, solving the problem of poor stability of PNP preparations at room temperature and achieving long-term stability and activity retention of nucleic acid molecules.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- SIRNAOMICS BIOPHARMACEUTICALS (SUZHOU) CO LTD
- Filing Date
- 2025-11-28
- Publication Date
- 2026-06-25
AI Technical Summary
Existing peptide nanoparticle (PNP) formulations have poor stability at room temperature, making it difficult to meet the requirements for long-term storage and transportation, and nucleic acid molecules are easily inactivated in the freeze-dried state.
A branched histidine-lysine polymer was used as a carrier, combined with trehalose or sucrose as a protective agent. The mass ratio of nucleic acid molecules, carrier and protective agent was optimized, and the nucleic acid lyophilized formulation was prepared by lyophilization after mixing in a microfluidic device.
It significantly improves the stability and activity of nucleic acid freeze-dried preparations, enabling long-term storage at room temperature and reducing the temperature requirements for transportation and storage.
Smart Images
Figure CN2025138361_25062026_PF_FP_ABST
Abstract
Description
A lyophilized nucleic acid formulation and its preparation method Technical Field
[0001] This invention belongs to the field of nucleic acid preparation technology, specifically relating to a nucleic acid freeze-dried preparation and its preparation method. Background Technology
[0002] After decades of research, the once simple concept that "RNA is merely a passive carrier of genetic information from DNA to protein" has undergone a revolutionary change. 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. Accompanying these great discoveries is the enormous potential of using RNA as a therapeutic approach, as it can lead to drugs for many targets previously considered "incurable" by traditional medicine. RNA therapy also eliminates the risk of inserted gene mutations compared to DNA-based gene therapy.
[0003] While RNA therapy holds great promise, one of the biggest challenges is safely delivering therapeutic RNA to the precise location within target cells. RNA molecules themselves are difficult to penetrate cell membranes due to their highly negatively charged nature and relatively bulky size. Furthermore, they are highly unstable, and typical storage conditions require extremely low temperatures (e.g., siRNA solids need to be stored at -20°C).
[0004] To overcome these limitations, research and development of various nanoparticle carriers is very active globally. Among them, peptide nanoparticles (PNPs) are another class of nanomaterials at the forefront of RNA drug delivery research. The advantages of peptide nanoparticles include scalability, diversity, precise tunability, reliability, biodegradability, and good drug release.
[0005] PNPs are nanoparticles formed by the self-assembly of HKP and nucleic acid molecules. PNPs play multiple roles in small nucleic acid drugs. HKP can form nanoparticles with small interfering nucleic acids (MIRNAs), which not only improves the stability of MIRNAs but also helps MIRNA drugs enter the cytoplasm of target cells, exerting a gene silencing effect. Because HKP is positively charged, it can complex with negatively charged RNA molecules mainly through electrostatic interactions, encapsulating RNA within the nanoparticles. PNP formulations encapsulate and immobilize nucleic acid molecules, effectively improving their stability (long-term storage temperature increases from -20°C to 4°C).
[0006] PNP preparations have been used in clinical practice. For example, STP705 and STP707 from Sinno Pharmaceuticals both use PNP technology.
[0007] Despite significant progress in PNP formulations, many challenges remain. One of these is how to further improve the stability of PNP formulations. While the stability of typical PNP formulations is greatly improved compared to naked nucleic acid molecules, the temperature (4°C) still places high demands on the transportation and storage of these drugs. Summary of the Invention
[0008] The purpose of this invention is to provide a nucleic acid freeze-dried preparation that is stable for long-term storage at room temperature and its preparation method.
[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0010] The first aspect of the present invention provides a nucleic acid lyophilized formulation, the nucleic acid lyophilized formulation comprising nucleic acid molecules, a carrier and a protectant, wherein the carrier is a branched histidine-lysine polymer, the protectant 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 protectant is 1:(35-150).
[0011] Preferably, the nucleic acid freeze-dried preparation is composed of the nucleic acid molecule, carrier, and protectant.
[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, or 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] Preferably, the mass ratio of the carrier to the protective agent is 1:(40-80). Too little protective agent will not achieve the desired effect, while too much will not further improve stability and will lead to a further increase in particle size and a decrease in the percentage of nucleic acid molecular weight.
[0016] More preferably, the mass ratio of the carrier to the protective agent is 1:(40-60).
[0017] Preferably, the nucleic acid molecule includes one or more of the following: messenger nucleic acid molecule (mRNA), small interfering nucleic acid molecule (siRNA), micro nucleic acid molecule (miRNA), small activating nucleic acid molecule (saRNA), antisense oligonucleotide molecule (ASO), or aptamer.
[0018] More preferably, the nucleic acid molecule is siRNA, comprising a sense strand and a reactive strand, the lengths of which are independently 17–28 nt, for example 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, and 28 nt. In some embodiments, the nucleic acid molecule comprises siRNA targeting TGF-β1 and / or siRNA targeting COX-2.
[0019] More specifically, the siRNA targeting TGF-β1 has the following nucleotide sequence:
[0020] Chain of Justice: 5'-CCCAAGGGCUACCAUGCCAACUUCU-3'
[0021] Antonym chain: 5'-AGAAGUUGGCAUGGUAGCCCUUGGG-3'.
[0022] More specifically, the COX-2-targeting siRNA has the following nucleotide sequence:
[0023] Chain of Justice: 5'-GGUCUGGUGCCUGGUCUGAUGAUGU-3'
[0024] Antonym chain: 5'-ACAUCAUCAGACCAGGCACCAGACC-3'.
[0025] 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.
[0026] More preferably, the mass ratio of the siRNA targeting TGF-β1 to 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, or 1.5:1.
[0027] 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.
[0028] Preferably, the carrier comprises HKP and / or HKP+H, wherein the HKP has the following structural formula:
[0029] K is lysine, and H is histidine;
[0030] The HKP+H structure is as follows:
[0031] K stands for lysine, and H stands for histidine.
[0032] The nucleic acid freeze-dried formulation of the present invention has a particle size of no more than 200 nm, a PDI of less than 1, and a zeta potential of 15–35 mV.
[0033] More preferably, the particle size of the lyophilized nucleic acid preparation does not exceed 180 nm, PDI < 0.8, and Zeta potential is 15–32 mV.
[0034] More preferably, the particle size of the nucleic acid freeze-dried preparation is no more than 150 nm, PDI < 0.5, and Zeta potential is 20-30 mV.
[0035] A second aspect of the present invention also provides a method for preparing the above-mentioned nucleic acid freeze-dried formulation, wherein an aqueous solution containing the nucleic acid molecules and an aqueous solution containing the carrier are first mixed using 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 formulation.
[0036] According to some specific embodiments of the present invention, the freeze-drying process is as follows:
[0037] (1) Pre-freezing: Pre-freeze at -1℃ to 1℃ for 5 to 15 minutes, cool down to -6℃ to -4℃ at a cooling rate of 0.2 to 0.4℃ / min, continue cooling down to -50℃ to -40℃ at a cooling rate of 0.6 to 1.5℃ / min, and pre-freeze at -50℃ to -40℃ for 5 to 8 hours.
[0038] (2) Drying: Dry at -50℃ to -40℃ for 30 to 50 min under a vacuum of 0.1 to 0.2 mbar, then heat to -30℃ to -20℃ at a heating rate of 0.6 to 1.5℃ / h, and dry at -30℃ to -20℃ for 68 to 78 h. Then heat to 20℃ to 30℃ at a heating rate of 45 to 55℃ / h, and dry at 20℃ to 30℃ for 8 to 12 h.
[0039] The present invention has at least the following beneficial effects:
[0040] When selecting sucrose and / or trehalose as a protective agent, this invention optimizes the mass ratio of branched histidine-lysine polymers, nucleic acid molecules, and the protective agent, significantly improving the storage stability of small interfering nucleic acid lyophilized formulations. This allows them to maintain nucleic acid molecule activity for extended periods at room temperature, reducing the temperature requirements for storage and transportation. This invention is of great significance for the development, application, and promotion of small interfering nucleic acid drugs. Attached Figure Description
[0041] Figure 1 is a schematic diagram of the mechanism of stabilizing nanoparticles in this invention (taking HKP and trehalose as examples). Detailed Implementation
[0042] To address the issues of poor stability, reduced nucleic acid purity, and easy inactivation in existing PNP formulations during room temperature storage and transportation, the inventors of this application have conducted extensive research and experimental exploration on formulation optimization. In preliminary experiments, the inventors attempted to improve the stability of the formulation by adjusting the ratio of the carrier to the nucleic acid molecules and by adding protective agents. However, none of these efforts effectively improved the stability of the formulation. For example, increasing the proportion of branched histidine-lysine polymer carriers improved the solution stability of the PNP formulation, but did not improve its long-term storage stability in the lyophilized state. Conversely, reducing the proportion of branched histidine-lysine polymer carriers significantly reduced both the solution stability and the long-term storage stability of the lyophilized formulation. Furthermore, adding lyophilization protectants (maltodextrin, microcrystalline cellulose, trehalose, glucose, dextran, sucrose, mannitol, sorbitol, xylitol, etc.) to existing PNP formulations did not significantly improve the room temperature storage stability of the PNP formulation. Through accidental operation, the inventors of this application discovered that reducing the proportion of branched histidine-lysine polymer carrier while adding an appropriate amount of trehalose or sucrose resulted in a formulation with unexpectedly high stability and good properties after lyophilization. Even after storage at room temperature for up to 24 months, it still exhibited good properties, high siRNA purity, and activity. Further exploration led to the development of the technical solution of this invention, as detailed below:
[0043] A nucleic acid freeze-dried formulation includes nucleic acid molecules, a carrier, and a protectant, wherein the carrier is a branched histidine-lysine polymer, and the protectant 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 protectant is 1:(35-150).
[0044] More preferably, the nucleic acid freeze-dried preparation comprises nucleic acid molecules, a carrier, and a protectant, wherein the carrier is composed of a branched histidine-lysine polymer, and more preferably, the mass ratio of the carrier to the nucleic acid molecules is (2-3):1, and even more preferably, the mass ratio of the carrier to the protectant is 1:(40-80).
[0045] Further investigation revealed that, taking HKP and trehalose as examples, the mechanism of the stable nanoparticles of the present invention is shown in Figure 1.
[0046] The following will provide further explanation of the technical solution, implementation process, and principles.
[0047] The technical solution and technical effects of the present invention will be further illustrated below with reference to specific embodiments and comparative examples.
[0048] Unless otherwise specified, the raw materials used in the following examples are commercially available.
[0049] The small interfering nucleic acids used in the following examples and comparative examples are a mixture of specific small interfering nucleic acids targeting TGF-β1 and specific small interfering nucleic acids targeting COX-2 in a mass ratio of 1:1.
[0050] The specific small interfering nucleic acid sequence targeting TGF-β1 is as follows:
[0051] Sense strand:5'-CCCAAGGGCUACCAUGCCAACUUCU-3' (SEQ ID NO: 1),
[0052] Antisense strand:5'-AGAAGUUGGCAUGGUAGCCCUUGGG-3' (SEQ ID NO: 2);
[0053] The specific small interfering nucleic acid sequence targeting COX-2 is as follows:
[0054] Sense strand:5'-GGUCUGGGCCCUGGUCUGAUGAUGU-3' (SEQ ID NO: 3),
[0055] Antisense strand: 5'-ACAUCAUCAGACCAGGCACCAGACC-3' (SEQ ID NO: 4).
[0056] The structural formula of HKP used in the following embodiments and comparative examples is as follows:
[0057] Unless otherwise specified, room temperature (RT) in the following examples refers to 20–25°C.
[0058] Unless otherwise specified, the experimental or testing methods involved in the following embodiments adopt conventional methods in the art.
[0059] Examples 1-14 and Comparative Examples 1-17 provide small interfering nucleic acid lyophilized formulations with different formulations. The components and ratios are shown in Table 1.
[0060] Table 1
[0061] Note: In Table 1, " / " indicates that it was not used, and the corresponding ratio is HKP:siRNA (mass ratio).
[0062] The preparation method of small-interference nucleic acid lyophilized formulation is as follows:
[0063] A 1 mg / mL siRNA solution was prepared using ultrapure water and siRNA. 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 were prepared using ultrapure water and HKP. A 50% protective agent aqueous solution (50 g / 100 mL) was prepared using ultrapure water and a protective agent. A suitable HKP solution was selected according to the mass ratio of HKP to siRNA in Table 1. This HKP solution was mixed with the siRNA solution at a volume ratio of 1:1 using microfluidic technology to obtain a PNP solution. Part of the PNP solution was lyophilized to obtain small interfering nucleic acid lyophilized formulations for Comparative Examples 1–5. 1 mL of the other PNP solution was added to a vial, and an appropriate amount of the 50% protective agent aqueous solution was added to the corresponding vial according to the preparation direction in Table 1. Lyophilization was then performed to obtain the small interfering nucleic acid lyophilized formulation. The lyophilization procedure is shown in Table 2.
[0064] Table 2
[0065] In Table 2, the times in steps (1), (4), (7), (9), and (11) are the times to maintain the corresponding temperature, and the times in steps (2), (3), (8), and (10) are the times to adjust from the temperature in the previous step to the temperature required in this step. Steps (5) and (6) are performed simultaneously. The vacuum degree “--” represents atmospheric pressure (one standard atmosphere, 1.013 × 10⁻⁶). 3 mbar).
[0066] Taking the small interfering nucleic acid lyophilized formulation (HKP:siRNA:trehalose = 1.5:1:60) of Example 1 as an example, its preparation method is as follows: 1 mg / mL siRNA solution and 1.5 mg / mL HKP solution are mixed by microfluidic technology. The total flow rate of mixing is 14 mL / min and the flow rate ratio is 1 / 1 to obtain a PNP solution with HKP / siRNA = 1.5. Then, it is dispensed into vials at 1 mL / vial. 0.06 mL of 50% trehalose aqueous solution is added to each vial. After mixing evenly, it is lyophilized according to the lyophilization procedure in Table 2.
[0067] For each example and comparative example, at least nine vials were prepared. Performance tests were performed on the day of preparation of the small interfering nucleic acid lyophilized formulation (day 0), after 6 months and 12 months of storage at room temperature. Each test was performed in triplicate, and the average value was taken. The testing method was as follows: 1 mL of water for injection was added to the vial to reconstitute the small interfering nucleic acid lyophilized formulation. Particle size (nm), PDI (polydispersity index), and Zeta potential (mV) were measured using a Zetasizer Nano ZS nanolaser particle size analyzer. The purity of siRNA was determined by anion exchange chromatography and calculated using the area normalization method. The test results are summarized in Table 3.
[0068] Table 3 Note: "NA" in Table 3 indicates that the product was not tested because it clearly did not meet the quality requirements.
[0069] The basic quality requirements for small interfering nucleic acid lyophilized preparations in Table 3 include: particle size not exceeding 200 nm, PDI < 1, Zeta potential 15–35 mV, maintaining powder form and remaining essentially unchanged in color.
[0070] As shown in Tables 1 and 3, when sucrose or trehalose is selected as the protectant, the optimized mass ratio of HKP, siRNA, and the protectant significantly improves the storage stability of small interfering nucleic acid lyophilized preparations. These preparations can maintain their properties and activity at room temperature for a long time, reducing the temperature requirements for storage and transportation of small interfering nucleic acid lyophilized preparations. This invention is of great significance for the development, application, and promotion of small interfering nucleic acid drugs.
Claims
1. A lyophilized formulation of a nucleic acid, characterized in that, The nucleic acid freeze-dried formulation comprises nucleic acid molecules, a carrier, and a protectant. The carrier is a branched histidine-lysine polymer, and the protectant 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 protectant is 1:(35-150).
2. The lyophilized formulation of nucleic acid according to claim 1, wherein, The nucleic acid freeze-dried preparation consists of the nucleic acid molecules, a carrier, and a protective agent.
3. The lyophilized formulation of nucleic acid of claim 1, wherein, The mass ratio of the vector to the nucleic acid molecule is (1.5–3.5):
1.
4. The lyophilized formulation of a nucleic acid according to claim 3, wherein, The mass ratio of the vector to the nucleic acid molecule is (2-3):
1.
5. The lyophilized formulation of a nucleic acid according to claim 1, wherein, The mass ratio of the carrier to the protective agent is 1:(40-120).
6. The lyophilized formulation of a nucleic acid according to claim 5, wherein, The mass ratio of the carrier to the protective agent is 1:(40-80).
7. The lyophilized formulation of a nucleic acid according to claim 1, wherein, The nucleic acid molecules include one or more of the following: messenger nucleic acid molecules, small interfering nucleic acid molecules, micro nucleic acid molecules, small activating nucleic acid molecules, antisense oligonucleotide molecules, or aptamers.
8. The lyophilized formulation of a nucleic acid according to claim 7, wherein, The nucleic acid molecule is siRNA, which includes a sense strand and a reaction strand, the lengths of which are independently 17–28 nt.
9. The lyophilized formulation of a nucleic acid according to claim 1, wherein, The nucleic acid molecules include siRNAs targeting TGF-β1 and / or siRNAs targeting COX-2.
10. The lyophilized formulation of a nucleic acid according to claim 9, wherein, The siRNA targeting TGF-β1 has the following nucleotide sequence: Chain of Justice: 5'-CCCAAGGGCUACCAUGCCAACUUCU-3' Antonym chain: 5'-AGAAGUUGGCAUGGUAGCCCUUGGG-3'; The COX-2-targeting siRNA has the following nucleotide sequence: Chain of Justice: 5'-GGUCUGGUGCCUGGUCUGAUGAUGU-3' Antonym chain: 5'-ACAUCAUCAGACCAGGCACCAGACC-3' 11. The lyophilized formulation of a nucleic acid according to claim 9, wherein, The nucleic acid molecule is a combination of siRNA targeting TGF-β1 and siRNA targeting COX-2.
12. The lyophilized formulation of a nucleic acid according to claim 11, wherein, The mass ratio of the siRNA targeting TGF-β1 to the siRNA targeting COX-2 is (0.5–1.5):
1.
13. The lyophilized formulation of a nucleic acid according to claim 1, wherein, The carrier comprises HKP and / or HKP + H, the HKP has the following structural formula: K is lysine, and H is histidine; The HKP+H structural formula is as follows: K stands for lysine, and H stands for histidine.
14. The method of preparing a lyophilized formulation of a nucleic acid according to any one of claims 1 to 13, wherein, First, an aqueous solution containing the nucleic acid molecules and an aqueous solution containing the carrier are mixed using a microfluidic device to form a nanoparticle solution. 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.
15. The method of claim 14, wherein the nucleic acid lyophilizate is prepared by, The freeze-drying process is as follows: (1) Pre-freezing: Pre-freeze at -1℃ to 1℃ for 5 to 15 minutes, cool down to -6℃ to -4℃ at a cooling rate of 0.2 to 0.4℃ / min, continue cooling down to -50℃ to -40℃ at a cooling rate of 0.6 to 1.5℃ / min, and pre-freeze at -50℃ to -40℃ for 5 to 8 hours. (2) Drying: Dry at -50℃ to -40℃ for 30 to 50 min under a vacuum of 0.1 to 0.2 mbar, then heat to -30℃ to -20℃ at a heating rate of 0.6 to 1.5℃ / h, and dry at -30℃ to -20℃ for 68 to 78 h. Then heat to 20℃ to 30℃ at a heating rate of 45 to 55℃ / h, and dry at 20℃ to 30℃ for 8 to 12 h.