WO2023207936A1 - 一种核酸-脂质纳米颗粒的冷冻干燥保护剂及其制备方法和应用 - Google Patents

一种核酸-脂质纳米颗粒的冷冻干燥保护剂及其制备方法和应用 Download PDF

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WO2023207936A1
WO2023207936A1 PCT/CN2023/090473 CN2023090473W WO2023207936A1 WO 2023207936 A1 WO2023207936 A1 WO 2023207936A1 CN 2023090473 W CN2023090473 W CN 2023090473W WO 2023207936 A1 WO2023207936 A1 WO 2023207936A1
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freeze
nucleic acid
drying
virus
application according
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French (fr)
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王浩猛
李明媛
严志红
贾琳
刘健
马文林
邱东旭
谢焱博
宇学峰
郁彭
朱涛
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康希诺生物股份公司
康希诺(上海)生物研发有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
    • A61J3/00—Devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms
    • A61J3/02—Devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms into the form of powders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of biomedicine and relates to a freeze-drying protective agent for nucleic acid-lipid nanoparticles and its preparation method and application.
  • the mRNA vaccine In the novel coronavirus pneumonia (COVID-19) epidemic, the mRNA vaccine has the advantages of safety, high immune response and low production cost. It has also received emergency approval for marketing in the United States and the European Union. In recent years, studies have found that transient protein expression induced by mRNA has great application value in many fields such as other infectious disease vaccines, cancer vaccines, cardiovascular diseases, protein replacement therapies, and genetic diseases. It can even achieve autonomous production of CAR through in vivo injection. -T effect. However, unlike the storage conditions of most vaccines (2-8°C), marketed mRNA vaccines have poor long-term stability and require ultra-low temperature storage (-20°C or even -70°C), and their validity period is less than 6 months. In the face of hundreds of millions of doses of mRNA vaccines that need to be stored, transported and distributed around the world, stability has become a bottleneck for its application.
  • the length of the mRNA chain is 1,000 to 5,000 bases.
  • RNase ribonuclease
  • LNP is used to deliver the mRNA, which not only protects the mRNA from instantaneous destruction by RNase in the body, but also breaks through the electrostatic repulsion barrier where both the mRNA and the cell membrane are negatively charged, enhancing delivery to antigen-presenting cells.
  • the long-term stability of mRNA-LNP injection is poor.
  • siRNA-LNP injection has good stability: Onpattro is very similar to the LNP in mRNA-1273, but has a 2-8°C and a 3-year validity period.
  • the siRNA-LNP injection constructed by Suzuki et al. was stored at 4°C for 1.5 years and had good stability.
  • siRNA has a small molecular weight and a double-stranded structure.
  • the chain length of mRNA is more than 100 times longer than siRNA and has a single-stranded structure.
  • the latter has a more unstable molecular structure and is more prone to hydrolysis.
  • LNP cannot protect the long-term stability of mRNA in injection solutions. role.
  • LNP consists of four main components: neutral phospholipids, cholesterol, polyethylene glycol (PEG)-lipids, and ionizable cationic lipids.
  • PEG polyethylene glycol
  • ionizable cationic lipids ionizable cationic lipids.
  • Viger Gra et al. found that two types of cores are possible: an amorphous core containing water pores surrounded by cationic lipids; or the lipids in the core can be evenly dispersed with small water pockets in the middle.
  • Freeze drying also known as sublimation drying, is a drying method in which water-containing materials are frozen below the freezing point according to the three-phase diagram of water, so that the water is converted into ice, and then the ice is converted into steam under a relatively high vacuum and removed.
  • lyophilized products have accounted for more than half of the biopharmaceuticals approved by the FDA and EMA.
  • Both are freeze-dried liposome preparations, with a shelf life of up to 36 months.
  • Dormitzer, Pfizer's head of viral vaccine research said the company intends to develop mRNA-LNP freeze-dried preparations.
  • liposomes are closed vesicles formed by phospholipid bilayers, and the liposomes used for freeze-drying are all Small molecule drugs;
  • mRNA-LNP has a shell-core structure, and the outer layer is only a phospholipid monolayer.
  • the mRNA contained in the core not only has a large molecular weight, but also has extremely high requirements for the integrity of the mRNA molecule.
  • mRNA-LNP needs to be freeze-dried in a vacuum.
  • Zhao et al. prepared a novel lipid-like nanoparticle lyophilized preparation for mRNA delivery. Although there was no significant difference in the extracellular activity of the mRNA rehydrated preparation and the original liquid, it did not have in vivo activity. The reason is unclear. Ai LX et al.
  • LNP LNP to encapsulate the S protein mRNA sequences of SARS-CoV-2 wild type, Delta strain and Omicron strain respectively, which can be stored at 4°C, 25°C and 40°C for 18 days.
  • Muramatsu et al. prepared mRNA-LNP freeze-drying The preparation can be stored at 4°C for 24 weeks, and there is no significant difference in biological activity in vivo and in vitro. It can be seen that the in vivo biological activities of the mRNA vaccines prepared under different freeze-drying conditions vary greatly, suggesting that different freeze-drying program curves and protective agents have a great impact on the performance of the preparations. However, the mechanism and rules of action have not been reported in the literature.
  • the freeze-drying process mainly includes two aspects: freeze-drying program curve and protective agent.
  • the freeze-drying program curve is a curve of temperature, vacuum degree and energy changing with time during the freeze-drying process. It generally includes three stages: pre-freezing, sublimation drying and desorption drying. Among them, the cooling temperature, heating rate and maintenance time all have an important impact on the appearance, moisture, particle size, etc. of the freeze-dried powder. Lyophilizing protectants, such as sucrose, trehalose, lactose and mannitol, can replace the hydrogen bonds between lipid molecules and water molecules to stabilize the LNP structure during the drying and dehydration process, and act as excipients.
  • freeze-drying protective agent makes it different from the molecular mechanism and micro-space effect of mRNA-LNP, which has a great impact on the LNP membrane structure, mRNA encapsulation rate, and mRNA integrity in the freeze-dried powder. Influence.
  • the impact of the freeze-drying program curve and protective agents on the microstructure, key physical and chemical properties and biological activity of mRNA-LNP freeze-dried powder needs to be further explored.
  • the freeze-drying process takes 30-100 hours from pre-freezing to analytical drying. On the one hand, it consumes energy. On the other hand, it also greatly increases the number of products. Amplify the time cost of production.
  • the preferred freeze-drying procedure in the invention patent CN 110714015 B is "the pre-freezing temperature is -50°C, and the temperature is maintained for 5 Hour.
  • the primary freeze-drying temperature is -40°C for 24 hours
  • the secondary freeze-drying temperature is 10°C for 17 hours
  • the vacuum degree during the freeze-drying process is 40 ⁇ bar.
  • It took a total of 46 hours.
  • the freeze-drying procedure of Muramatsu et al. to prepare the mRNA-LNP lyophilized preparation took a total of 84 hours.
  • the first purpose of the present invention is to improve the storage stability of nucleic acid-lipid nanoparticles.
  • the storage conditions can be reduced to refrigeration (2-8°C), and on the other hand, the validity period can be extended. It also rehydrates quickly.
  • the second object of the present invention is to significantly shorten the total duration of the freeze-drying process, improve efficiency, and reduce production energy and time costs.
  • the third purpose of the present invention is to screen out a specific type and dosage of freeze-drying protective agent for nucleic acid-lipid nanoparticles, fully maintain the total amount of nucleic acid, encapsulation rate and integrity of its freeze-dried rehydrated preparation, and have non-destructive properties. In vivo and in vitro biological activities.
  • the fourth object of the present invention is to break through the "empirical" development model of the freeze-drying process and establish an evaluation method for key physical and chemical properties.
  • lipid nanoparticle refers to particles having at least one nanometer-scale dimension, which comprise at least one lipid.
  • the nucleic acid-lipid nanoparticles (LNP) of the present invention include nucleic acid, neutral lipids, protonatable cationic lipids, sterol lipids, and PEG-lipids.
  • nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and includes DNA, RNA and hybrids or derivatives thereof.
  • lipid herein refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water but soluble in many organic solvents.
  • protonatable cationic lipid herein refers to lipid molecules that are capable of being positively charged in certain environments, such as ALC-0315.
  • neutral lipid refers to uncharged, non-phosphoglyceride lipid molecules.
  • PEG-lipid polyethylene glycol lipid
  • PEG-lipid polyethylene glycol lipid
  • vaccine refers to a composition suitable for application to animals, including humans, which upon administration induces an immune response of sufficient strength to minimally assist in the prevention, amelioration or cure of clinical disease resulting from infection by a microorganism.
  • delivery system in the present invention refers to a preparation or composition that regulates the distribution of biologically active ingredients in space, time and dosage in the body.
  • nucleic acid-lipid nanoparticles include 25-75% protonatable cationic lipids, 5-20% neutral lipids, 0-50% sterol lipids and 1-5% PEG based on molar percentages Lipids.
  • the invention provides a freeze-drying protective agent for nucleic acid-lipid nanoparticles, including the following components: sucrose, trehalose, and the mass ratio of sucrose to trehalose is 5-15 :5-11.
  • the freeze-drying protective agent used for nucleic acid-lipid nanoparticles of the present invention includes: one or more of mannitol, glucose or lactose.
  • the mass ratio of sucrose, trehalose, mannitol or glucose or lactose in the prescription of the freeze-drying protective agent of the present invention is 5-15:5-11:1-5.
  • the present invention provides a method for preparing a freeze-drying agent for nucleic acid-lipid nanoparticles. According to corresponding proportions, sucrose and trehalose are dissolved in nuclease-free water.
  • the present invention provides a method for preparing a freeze-drying agent for nucleic acid-lipid nanoparticles. According to the corresponding ratio, sucrose, trehalose, mannitol or glucose or lactose are dissolved in nuclease-free water.
  • the invention provides the use of freeze-drying protective agents for nucleic acid-lipid nanoparticles in the preparation of drugs.
  • nucleic acid-lipid nanoparticles are added to the solution of the lyoprotectant for freeze-drying.
  • freeze-drying includes the following steps: pre-freezing stage, sublimation drying, and desorption drying.
  • the pre-freezing stage the temperature is -80°C to -50°C, and the pre-freezing time is 2 to 6 hours.
  • the sublimation drying stage the temperature is -60°C ⁇ 0°C, the end temperature is -5°C ⁇ 0°C, and the vacuum pressure is controlled ⁇ 10Pa; preferably, the sublimation drying stage is divided into 2 to 8 sections with a gradient temperature rise, and each section is heated by 5 ⁇ 30°C, each stage maintenance time is 0.5 ⁇ 10 hours, vacuum pressure control ⁇ 10Pa.
  • the desorption drying stage the temperature is 0°C to 35°C, the end temperature is 30°C to 35°C, the operation time is 2 to 8 hours, and the vacuum pressure is controlled to ⁇ 10Pa.
  • the nucleic acid-lipid nanoparticles include ssDNA, dsDNA, mRNA, lncRNA, siRNA, saRNA, shRNA, ASO (antisense oligonucleotide), plasmid, circRNA, circDNA, miRNA, CRISPR-Cas, ployI:C , SamRNA, 5'-pppRNA.
  • the drug is a vaccine
  • the vaccine is used to prevent cancer, viral infection, bacterial infection, fungal infection
  • the virus is selected from: norovirus, Ebola virus, coronavirus , cytomegalovirus, dengue virus, Zika virus, coxsackie virus, enterovirus, hepatitis virus, herpes simplex virus, human papilloma virus, influenza virus, Marburg virus, measles virus, polio virus, rabies virus, Rotavirus, measles virus.
  • the freeze-drying agent is added to the vaccine in an amount of 5-20% (w/w%).
  • the drug is an oral formulation, intramuscular injection formulation, intravenous injection formulation or inhalation formulation.
  • the inhalation preparation is an aerosol inhalation agent or a dry powder inhalation agent.
  • the present invention has the following beneficial effects:
  • the present invention can shorten the total length of the freeze-drying process of nucleic acid-lipid nanoparticles to 8 to 18 hours, significantly reducing energy consumption and the time cost of product scale-up production. Moreover, the freeze-dried nucleic acid-lipid nanoparticles prepared by the freeze-drying process of the present invention can be rehydrated quickly (within 10s), and the total nucleic acid content, encapsulation rate and nucleic acid integrity are high.
  • freeze-drying can reduce or even remove the water-containing microenvironment in the system, which can inhibit the nucleic acid hydrolysis reaction catalyzed by nucleases, significantly improve the long-term stability of nucleic acid-lipid nanoparticles, extend the validity period, and increase the storage temperature to 2 ⁇ 8°C, significantly reducing the cost and efficiency of storage and transportation.
  • Figure 1 The particle size distribution diagram of the freeze-dried and rehydrated preparation of Example 1-1 measured three times.
  • Figure 2 is a SEM scanning image of the sample obtained in Example 1-1.
  • Figure 3 is a SEM scanning image of the samples obtained in Examples 1-13.
  • Figure 4 is a SEM scanning image of the samples obtained in Examples 1-14.
  • Figure 5 is a SEM scanning image of the samples obtained in Examples 1-15.
  • Figure 6 is a SEM scanning image of the samples obtained in Examples 1-16.
  • Figure 7 is a SEM scanning image of the sample obtained in Examples 1-17.
  • Figure 8 is a SEM scanning image of the samples obtained in Examples 1-18.
  • Figure 9 is a SEM scanning image of the sample obtained in Examples 1-19.
  • Figure 10 is a SEM scanning image of the samples obtained in Examples 1-20.
  • Figure 11 is a SEM scanning image of the sample obtained in Examples 1-21.
  • Figure 12 is an SEM scanning image of the sample obtained in Comparative Example 1-1.
  • Figure 13 is the TEM micromorphological image of the model sample.
  • Figure 14 is a TEM micromorphological image of the sample obtained in Example 1-1.
  • Figure 15 is a TEM micromorphological image of the sample obtained in Examples 1-4.
  • Figure 16 is a TEM micromorphological image of the sample obtained in Examples 1-8.
  • Figure 17 is a TEM micromorphological image of the sample obtained in Examples 1-12.
  • Figure 18 is a TEM micromorphological image of the sample obtained in Comparative Examples 1-10.
  • Figure 19A is a test chart of model sample mRNA integrity analysis.
  • Figure 19B is a test chart of the mRNA integrity analysis of the sample obtained in Example 2-1.
  • Figure 19C is a picture of the mRNA integrity analysis test of the sample obtained in Example 2-5.
  • Figure 20 Expression of protein transfected into mRNA-LNP cells.
  • the mRNA encoding the SARS-CoV-2 spike protein (S-mRNA, approximately 4000 bp) was used as the model mRNA, and the mRNA lipid nanoparticles (i.e., mRNA-LNP) without adding protective agents and not freeze-dried were used as the model sample.
  • S-mRNA SARS-CoV-2 spike protein
  • mRNA-LNP mRNA lipid nanoparticles
  • the preparation process is as follows: dilute the mRNA stock solution with sodium acetate buffer to a concentration of 135 ⁇ g/ml, and prepare the lipid according to the protonatable cationic lipid ALC-0315: DSPC: cholesterol: DMG-PEG 2000 molar ratio of 49:10:39.5:1.5 Mix the solution; after completing the encapsulation on the nanomedicine manufacturing equipment, change the liquid by ultrafiltration and collect the sample.
  • the mRNA encoding the SARS-CoV-2 spike protein (S-mRNA, approximately 4000 bp) was used as the model mRNA, and the mRNA liposome (i.e., mRNA-Lip) without adding a protective agent and not freeze-dried was used as the liposome sample.
  • S-mRNA SARS-CoV-2 spike protein
  • mRNA liposome i.e., mRNA-Lip
  • the preparation process is as follows: using a DOTAP:DOPE molar ratio of 1:1 as the liposome membrane material, using a film dispersion method to prepare cationic liposomes, then mixing and encapsulating them with mRNA, and adjusting them to be consistent with the concentration of mRNA in the model sample.
  • Example 1-1 to 1-12 and Comparative Examples 1-1 to 1-28 were used as the protective agent and model sample lipid nanoparticles (i.e., mRNA- LNP) and mix them evenly, and distribute them into vials;
  • Comparative Example 1-29 is to mix the protective agent of Example 1-1 and the liposome sample (i.e., nucleic acid-Lip) evenly, that is, replace the model sample with the liposome sample. Dispense into vials.
  • Measurement of particle size, potential and rehydration time Take model samples and samples of Examples 1-1 to 1-21 and Comparative Examples 1-1 to 1-29 respectively (add 0.5 mL of non-alkali to the freeze-dried samples of Examples and Comparative Examples). Rehydrate with nuclease water and record the time required for rehydration), then add 1 mL of nuclease-free water, and measure the particle size, polydispersity coefficient and Zeta potential with a Malvern particle size analyzer at 25 ⁇ 1°C. Measure three times in parallel, and take For average values, see Table 2.
  • the particle size of Examples 1-1 to 12 increased by ⁇ 25 nm.
  • the particle size distribution of the freeze-dried powder obtained by the freeze-drying process of Example 1-1 after rehydration is shown in Figure 1.
  • the particle size of Comparative Examples 1-1 to 1-28 increased by about 60-320 nm, indicating that the nanostructure of the nucleic acid-lipid nanoparticles has been damaged.
  • the combination of sucrose and trehalose as a freeze-drying protective agent has more protective effects than the two protective agents used alone, and the combination of sucrose, trehalose and mannitol has the best effect.
  • the freezing stress can be divided into four situations: the formation of dendritic ice crystals, increase in ion concentration, change in pH value and phase separation. Sucrose, trehalose and mannitol are mixed in an appropriate proportion, and mRNA-LNP is added in an appropriate amount for freeze-drying.
  • the powder cake is highly rigid, not easy to disintegrate, can resist various stress damages, and has good stability; on the other hand, it has high porosity and is beneficial During the freeze-drying process, the water evaporates quickly. During the rehydration process, the specific surface area for wetting with water molecules is large, so the effect of rapid rehydration can be achieved.
  • Example 1-1 Although the protective agent in Example 1-1 is the same as that in Comparative Example 1-29, compared with the liposome sample, the particle size of Comparative Example 1-29 increased by more than 75 nm, indicating that the protective agent system has nucleic acid-LNP specificity, that is, it is suitable for nucleic acids. -LNP formulations, not suitable for nucleic acids -Lip.
  • the embodiment has a "ginger-shaped" wrapped scaffold microstructure, while in the comparative example, a scaffold structure is not formed between the LNP and the protective agent, and the protective agent can only play a role in dispersing filling and reducing stress damage.
  • Example 1-1, Example 1-4, Example 1-8, Example 1-12, and Comparative Example 1-10 Dilute the model sample and Example 1-1, Example 1-4, Example 1-8, Example 1-12, and Comparative Example 1-10 by adding an appropriate amount of nuclease-free water, and then add dropwise dilution on the copper grid.
  • For the sample use filter paper to absorb the excess sample from the edge of the copper mesh, then add 2% phosphotungstic acid dropwise for staining for 5 seconds, use filter paper to absorb the excess phosphotungstic acid from the edge of the copper mesh, and use a transmission electron microscope to scan and image.
  • the results are shown in the attached figure. 13-18, it can be seen that the examples have similar particle size distribution and micromorphology compared to the comparative examples and the model samples.
  • Stability storage conditions 2-8°C, 1 month.
  • the model samples, Examples 2-1 to 6, and Comparative Examples 2-1 to 6 were respectively measured at 0 o'clock and 1 month, the total amount of mRNA, free mRNA concentration, encapsulation rate and mRNA integrity of each group.
  • the results are shown in Table 4.
  • Encapsulation efficiency (total amount of mRNA - free amount of mRNA)/total amount of mRNA * 100%
  • Determination of mRNA integrity First dilute the mRNA-LNP sample and denature it at 70°C. Prepare Dye-Gel gel and inject it into the gel press. Then, add the mRNA marker, ladder and sample respectively. The Agilent 2100 bioanalytical system was used to efficiently determine the sample mRNA integrity spectrum based on the principle of gel electrophoresis. The mRNA integrity measurement patterns of the model sample, Example 2-1 and Example 2-5 at 0 point are shown in Figure 19 (A/B/C).
  • the gentle multi-step heating method in the freeze-drying process is more conducive to protecting the nanostructure of nucleic acid-lipid nanoparticles, improving the mRNA encapsulation rate and integrity, and improving the stability of the freeze-dried preparation.
  • HEK293 cells were used as in vitro protein expression model cells.
  • the cells were plated in a 6-well plate at a cell density of 1 ⁇ 10 6 cells/mL and cultured for 24 h.
  • Example 1-1, Example 1-4, Example 1-8 and Example 1-12 were added without Re-dissolve the nuclease in water, then take the model sample and add it to the wells in order, the amount added is 20 ⁇ L, mix well, transfect for 4 hours, and change the medium (including serum).
  • digest the cells with PBS transfer them to a centrifuge tube and centrifuge (2500r/min, 5min).
  • the condensing gel solidified; put the device into the electrophoresis tank, add electrophoresis solution (1 ⁇ SDS-PAGE buffer) to cover the platinum wire, pull out the comb, and add Example 1-1, Example 1-4, Examples 1-8 and 1-12, protein expression samples of model samples; electrophoresis (100V, 300mA), observe the position of bromophenol blue and marker during electrophoresis, wait until it reaches the bottom of the gel Then stop; take out the gel after electrophoresis, separate it according to the dividing line between the stacking gel and the separation gel, remove the stacking gel, keep the separation gel, soak the separation gel and filter paper in 1 ⁇ bloting buffer, and soak the PVDF membrane in anhydrous methanol Soak to activate; put 3 layers of filter paper, 1 layer of PVDF membrane, separation gel, and 3 layers of filter paper on the film transfer apparatus in the order and drive out the bubbles, turn on the power, 15V (5V to 15V ladder (increased pressure
  • Skim milk powder put the membrane into a 15mL centrifuge tube containing the primary antibody (diluted with 5% skim milk powder according to the instructions), incubate at 4°C overnight, and wash away the excess primary antibody with 1 ⁇ PBST; remove the PVDF that has blocked the primary antibody
  • the membrane was placed in a 15mL centrifuge tube containing secondary antibody (diluted with 5% skimmed milk powder according to the instructions), and incubated at room temperature for 1 hour (be careful to avoid light). After washing away the excess secondary antibody, the Odyssey infrared laser imaging system was used to detect and analyze.
  • mice BALB/c mice were taken, and the rehydration preparations and model samples of Example 1-1, Example 1-4, Example 1-8 and Example 1-12 were used for comparative studies.
  • the mice were randomly divided into 5 groups.
  • Example 1-1, Example 1-4, Example 1-8 and Example 1-12 can all induce sufficient immune responses in vivo, and the immune response level of Examples 1-8 even exceeds that of the model sample on the 28th day after the initial immunization, indicating that the freeze-drying process designed in the present invention can maintain or even improve mRNA-LNP immune effects in the body.

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Abstract

本发明基于冷冻干燥工艺的物理化学原理，对于核酸-脂质纳米颗粒的冷冻干燥条件，即冷冻干燥程序和冻干保护剂进行了系统研究，优选并设计了适宜于核酸-脂质纳米颗粒的高效冷冻干燥方法。本发明可将核酸-脂质纳米颗粒冷冻干燥工艺总时长缩短至8-18小时，显著降低能源消耗和产品放大生产的时间成本，冻干的核酸-脂质纳米颗粒，复水迅速(10s以内)，核酸总量、包封率及核酸完整性高，除此之外，冻干复水后制剂的细胞转染效率与和未冻干核酸-脂质纳米颗粒体原液无显著性差异，且体内免疫应答高，甚至超过未冻干核酸-脂质纳米颗粒体原液。

Description

一种核酸-脂质纳米颗粒的冷冻干燥保护剂及其制备方法和应用 技术领域
本发明属于生物医药技术领域，涉及一种核酸-脂质纳米颗粒的冷冻干燥保护剂及其制备方法和应用。
背景技术
新型冠状病毒肺炎(COVID-19)疫情中，mRNA疫苗具有安全、免疫应答高和生产成本低的优势，同时获得美国和欧盟紧急获批上市。近年来，研究发现由mRNA诱导的瞬时蛋白表达在其他传染病疫苗、癌症疫苗、心血管疾病、蛋白质替代疗法和遗传病等多领域均具有巨大的应用价值，甚至能够通过体内注射实现自主产生CAR-T效应。然而，与绝大多数疫苗的储存条件(2-8℃)不同，已上市mRNA疫苗长期稳定性差，需超低温保存(-20℃，甚至-70℃)，且有效期低于6个月。在面临数亿剂mRNA疫苗需要储存，运输和分发到世界各地的情况下，稳定性成为其应用的瓶颈问题。
首先，mRNA链长为1000至5000个碱基长度，早期研究中已发现裸mRNA会被核糖核酸酶(RNase)迅速水解，而RNase无处不在，体内外均有大量附着，需要特殊处理才能去除。除此之外，mRNA长链上仅一个变化(键断裂或碱基的氧化)即会终止翻译，故mRNA疫苗中mRNA分子的完整性至关重要。
在mRNA疫苗中为了提高mRNA体内作用效力，以LNP包载递送mRNA，不仅保护mRNA避免被体内RNase瞬时破坏，而且突破mRNA与细胞膜均带负电荷的静电排斥屏障，增强向抗原递呈细胞的递送。然而，mRNA-LNP注射液长期稳定性不佳，与其相反，siRNA-LNP注射液稳定性良好：Onpattro与mRNA-1273中的LNP十分相似，却具有2-8℃，3年有效期。除此之外，Suzuki等构建的siRNA-LNP注射液4℃放置1.5年，稳定性良好。siRNA分子量小且为双链结构，mRNA链长超过siRNA 100倍以上且为单链结构，后者分子结构更不稳定，更易发生水解，上述研究表明LNP在注射液中不能起到保护mRNA长期稳定的作用。
LNP由四种主要成分组成：中性磷脂、胆固醇、聚乙二醇(PEG)-脂质和可电离阳离子脂质。研究发现mRNA-LNP为核-壳结构，具有表面层和一个无定形、各向同性的核心。Viger Gra等利用NMR波谱发现两种类型的核心是可能的：无定形核包含被阳离子脂质包围的水孔；或核心中的脂质可以均匀分散，中间有小水袋。Arteta等和Sebastiani等发现中性 磷脂和PEG脂质以及部分可电离阳离子脂质和胆固醇位于LNP表面，而可电离阳离子脂质、胆固醇、水和mRNA位于核心区域。研究表明LNP核心含水24％，mRNA位于被阳离子脂质包围的水柱内。
冷冻干燥，又称升华干燥，根据水的三相图将含水物料冷冻到冰点以下，使水转变为冰，然后在较高真空下将冰转变为蒸气而除去的干燥方法。近年来，冻干产品占FDA和EMA批准生物药品的一半以上。在纳米制剂领域，和均为冻干脂质体制剂，二者保质期可达36个月。辉瑞病毒疫苗研究主管Dormitzer表示公司有意开发mRNA-LNP冻干制剂。然而，目前关于mRNA-LNP冻干条件的研究很少，虽然均为纳米制剂，但脂质体是磷脂双分子层形成的闭合囊泡，且用于冻干的脂质体包载的皆为小分子药物；mRNA-LNP为壳-核结构，外层仅为磷脂单分子层，核心包载的mRNA不仅分子量大，且对于mRNA分子的完整性要求极高，mRNA-LNP需要在真空冷冻干燥的高强度压力变化过程中，保持其磷脂层和纳米结构不被破坏，保证mRNA的包载状态不发生变化，并在复水重构后恢复原液粒径分布及mRNA包封率，这些都具有很高的挑战性。Zhao等制备了一种新型类脂质纳米颗粒用于mRNA递送的冻干制剂，虽然mRNA复水制剂与原液体外细胞活性无显著差异，但却不具备体内活性，尚不清楚原因。Ai LX等以LNP分别包载SARS-CoV-2野生型、Delta株和Omicron株的S蛋白mRNA序列，可于4℃、25℃和40℃储存18天，Muramatsu等制备了mRNA-LNP冻干制剂，可于4℃储存24周，且体内外生物活性无显著差异。可见不同冻干条件制备的mRNA疫苗体内生物活性差异极大，提示不同的冻干程序曲线和保护剂对于制剂性能有很大影响，但对其中的作用机理和规律尚未见文献报道。
冷冻干燥工艺主要包括冻干程序曲线和保护剂两方面内容。冻干程序曲线是冻干过程中的温度、真空度及能量等随时间变化的曲线，一般包括三个阶段：预冻，升华干燥和解吸干燥。其中，冷却温度、升温速率及维持时长等均对于冻干粉的外观、水分、粒径等有重要影响。冻干保护剂，如蔗糖、海藻糖、乳糖和甘露醇等，可于干燥脱水过程中取代脂质分子与水分子间的氢键来稳定LNP结构，并起到赋形剂的作用。冻干保护剂种类、用量及添加顺序，均使其与mRNA-LNP的分子作用机制及微观空间作用不同，对冻干粉中的LNP膜结构，mRNA包封率，mRNA完整性等有很大影响。综上所述，冻干程序曲线和保护剂对mRNA-LNP冻干粉的微观结构、关键理化性质及生物活性的影响，亟需深入探究。
现有技术中的冻干工艺关键参数常为“经验式”应用，一般冷冻干燥工艺从预冻至解析干燥结束，需要30-100小时，耗费能源是一个方面，另一方面也大大增加了产品放大生产的时间成本，如发明专利CN 110714015 B中优选的冻干程序为“预冻温度为-50℃,温度保持5 小时。一次冻干温度为-40℃24小时，二次冻干温度为10℃保持17小时，冻干过程真空度为40μbar。”共耗时46小时。Muramatsu等制备mRNA-LNP冻干制剂的冷冻干燥程序共耗时84小时。
发明内容
本发明的第一目的在于提高核酸-脂质纳米颗粒的储存稳定性，一方面将储存条件降低为冷藏即可(2-8℃)，另一方面可延长有效期。同时复水迅速。
本发明的第二目的在于可显著缩短冻干程序总时长，提高效率，降低生产的能源和时间成本。
本发明的第三目的在于针对核酸-脂质纳米颗粒筛选出特定种类及用量的冻干保护剂，充分保持其冻干复水制剂的核酸总量、包封率和完整性，并具有无损的体内外生物活性。
本发明的第四目的在于本发明突破冷冻干燥工艺“经验式”的开发模式，建立了对关键理化性质评价方法。
本发明术语“脂质纳米颗粒”是指具有至少一个纳米量级尺寸的颗粒，其包含至少一种脂质。
本发明所述的核酸-脂质纳米颗粒(LNP)，成分包括核酸、中性脂质、可质子化阳离子脂质、甾醇脂质、PEG-脂质。
本发明术语“核酸”是指呈单链或双链形式的含有至少两种脱氧核糖核苷酸或核糖核苷酸的聚合物，并且包括DNA、RNA及其杂交物或衍生物。
本发明术语“脂质”是指一组有机化合物，其包括但不限于脂肪酸的酯，并且通常以难溶于水但可溶于许多有机溶剂为特征。
本发明术语“可质子化阳离子脂质”是指在某些环境中能够带正电的脂质分子，例如ALC-0315。
本发明术语“中性脂质”术语是指不带电荷的、非磷酸甘油酯的脂质分子。
本发明术语“PEG-脂质(聚乙二醇脂质)”是指包含脂质部分和聚乙二醇部分的分子。
本发明术语“疫苗”是指适合于应用于动物(包括人)的组合物，在施用后诱导免疫应答，其强度足以最低限度地帮助预防、改善或治愈起因于由微生物感染的临床疾病。
本发明术语“递送系统”是指调控生物活性成分在空间、时间及剂量在生物体内分布的制剂或组合物。
具体地，核酸-脂质纳米颗粒，LNP按照摩尔百分比计，包括25～75％可质子化阳离子脂质、5～20％中性脂质、0～50％甾醇脂质和1～5％PEG脂质。
为了实现本发明的上述目的，特采用以下技术方案：
根据本发明的一个方面，本发明提供了一种用于核酸-脂质纳米颗粒的冷冻干燥保护剂，包括以下组分：蔗糖、海藻糖，所述蔗糖与海藻糖的质量比为5-15：5-11。
本发明用于核酸-脂质纳米颗粒的冷冻干燥保护剂，包括：甘露醇、葡萄糖或乳糖中的一种或多种。
根据本发明的一个方面，本发明冷冻干燥保护剂的处方中蔗糖、海藻糖、甘露醇或葡萄糖或乳糖的质量比为5-15：5-11：1-5。
根据本发明的一个方面，本发明提供用于核酸-脂质纳米颗粒的冷冻干燥剂的制备方法，按照相应配比，将蔗糖、海藻糖溶解在无核酸酶水中。
根据本发明的一个方面，本发明提供用于核酸-脂质纳米颗粒的冷冻干燥剂的制备方法，按照相应配比，将蔗糖、海藻糖、甘露醇或葡萄糖或乳糖溶解在无核酸酶水中。
根据本发明的一个方面，本发明提供用于核酸-脂质纳米颗粒的冷冻干燥保护剂在制备药物中的应用。
优选地，将核酸-脂质纳米颗粒加入到所述冻干保护剂的溶液中进行冷冻干燥。
优选地，冷冻干燥包括以下步骤：预冻阶段、升华干燥、解吸干燥。
优选地，预冻阶段：温度为-80℃～-50℃，预冻时间为2～6小时。
优选地，升华干燥阶段：温度为-60℃～0℃，终点温度-5℃～0℃，真空压力控制≤10Pa；优选地，升华干燥阶段分2～8段呈梯度升温，每段升温5～30℃，每段维持时间0.5～10小时，真空压力控制≤10Pa。
优选地，解吸干燥阶段：温度为0℃～35℃，且终点温度30～35℃，运行时间2～8小时，真空压力控制≤10Pa。
优选地，核酸-脂质纳米颗粒中包括ssDNA，dsDNA，mRNA，lncRNA，siRNA，saRNA，shRNA，ASO(反义寡核苷酸)，质粒，circRNA，circDNA，miRNA，CRISPR-Cas，ployI:C，SamRNA，5’-pppRNA。
优选地，药物为疫苗，更优选的，疫苗用于预防癌症、病毒感染、细菌感染、真菌感染的疫苗；更优选的，所述的病毒选自：诺如病毒、埃博拉病毒、冠状病毒、巨细胞病毒、登革热病毒、寨卡病毒、柯萨奇病毒、肠病毒、肝炎病毒、单纯疱疹病毒、人乳头瘤病毒、流感病毒、马尔堡病毒、麻疹病毒、脊髓灰质炎病毒、狂犬病病毒、轮状病毒、麻疹病毒。
优选地，冷冻干燥剂在所述疫苗中加入量为5-20％(w/w％)。
优选地，药物为口服制剂、肌肉注射制剂、静脉注射制剂或吸入制剂。
优选地，吸入制剂为雾化吸入剂或干粉吸入剂。
与现有技术相比，本发明具有如下有益效果：
本发明可将核酸-脂质纳米颗粒冷冻干燥工艺总时长缩短至8～18小时，显著降低能源消耗和产品放大生产的时间成本。且本发明冷冻干燥工艺制备的冻干核酸-脂质纳米颗粒，复水迅速(10s以内)，核酸总量、包封率及核酸完整性高，除此之外，冻干复水后制剂的细胞转染效率与和未冻干核酸-脂质纳米颗粒体原液无显著性差异，且体内免疫应答高，甚至超过未冻干核酸-脂质纳米颗粒体原液。除此之外，通过冷冻干燥降低甚至去除体系内的含水微环境，可以抑制核酸酶催化的核酸水解反应，显著提高核酸-脂质纳米颗粒的长期稳定性，延长有效期，提高保存温度为2～8℃，显著降低储存和运输的成本和效率。除此之外，将核酸-脂质纳米颗粒的制剂相态由液态转变为固态，为其新型固体制剂的开发提供可能，具有重要意义。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1实施例1-1冻干复水制剂测定三次的粒径分布图。
图2为实施例1-1所得样品SEM扫描成像。
图3为实施例1-13所得样品SEM扫描成像。
图4为实施例1-14所得样品SEM扫描成像。
图5为实施例1-15所得样品SEM扫描成像。
图6为实施例1-16所得样品SEM扫描成像。
图7为实施例1-17所得样品SEM扫描成像。
图8为实施例1-18所得样品SEM扫描成像。
图9为实施例1-19所得样品SEM扫描成像。
图10为实施例1-20所得样品SEM扫描成像。
图11为实施例1-21所得样品SEM扫描成像。
图12为对比例1-1所得样品SEM扫描成像。
图13为模型样品的TEM微观形态图。
图14为实施例1-1所得样品的TEM微观形态图。
图15为实施例1-4所得样品的TEM微观形态图。
图16为实施例1-8所得样品的TEM微观形态图。
图17为实施例1-12所得样品的TEM微观形态图。
图18为对比例1-10所得样品的TEM微观形态图。
图19A为模型样品mRNA完整性分析测试图。
图19B为实施例2-1所得样品mRNA完整性分析测试图。
图19C为实施例2-5所得样品mRNA完整性分析测试图。
图20 mRNA-LNP细胞转染蛋白表达量。
图21初次免疫14天后免疫效应结果。
图22初次免疫28天(二次免疫14天)后免疫效应结果。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下面结合优选实施例进一步说明本发明的技术方案和有益效果。
模型样品：
以编码SARS-CoV-2棘突蛋白的mRNA(S-mRNA，约4000bp)为模型mRNA，以未加入保护剂、未冷冻干燥的mRNA脂质纳米颗粒(即mRNA-LNP)为模型样品。
制备过程如下：醋酸钠缓冲液稀释mRNA原液至浓度为135μg/ml，按照可质子化阳离子脂质ALC-0315：DSPC：胆固醇：DMG-PEG 2000摩尔比为49：10：39.5：1.5配制脂质混合溶液；在纳米药物制造设备上完成包封后，超滤换液，收集样品。
脂质体样品：
以编码SARS-CoV-2棘突蛋白的mRNA(S-mRNA，约4000bp)为模型mRNA，以未加入保护剂、未冷冻干燥的mRNA脂质体(即mRNA-Lip)为脂质体样品。
制备过程如下：以DOTAP：DOPE摩尔比为1:1为脂质体膜材，利用薄膜分散法制备阳离子脂质体，再与mRNA混合包载，调整至与模型样品中mRNA浓度一致。
实施例1 冻干保护剂的制备
按照表1配方在溶剂中配制成冻干保护剂，其中溶剂为无核酸酶水，以0.22μm无菌滤膜过滤除菌。以冻干保护剂添加量为10％(w/w)，实施例1-1～1-12及对比例1-1～1-28样品为保护剂与模型样品脂质纳米颗粒(即mRNA-LNP)混合均匀，分装至西林瓶中；对比例1-29为将实施例1-1的保护剂与脂质体样品(即核酸-Lip)混合均匀，即将模型样品替换为脂质体样品分装至西林瓶中。
表1冻干保护剂的处方


取实施例1-1～1-21及对比例1-1～1-29样品500μl至3ml西林瓶中，进行冷冻干燥。冷冻干燥程序：预冻阶段：-60℃4小时；升华干燥阶段：-45℃2小时，-25℃2小时，-5℃2小时，全程真空压力控制≤10Pa；解吸干燥阶段：30℃，2小时，全程真空压力控制≤10Pa。
粒径、电位及复水时间测定：分别取模型样品以及实施例1-1～1-21、对比例1-1～1-29样品(向实施例及对比例冻干样品加入0.5mL的无核酸酶水复水，记录复水所需时间)，再补充1mL无核酸酶水，于马尔文粒度仪在25±1℃进行粒径、多分散系数和Zeta电位的测量，平行测量三次，取平均值，见表2。
表2粒径和电位


可见实施例1-1～12与模型样品相比，粒径增长<25nm，其中，实施例1-1的冷冻干燥工艺所得冻干粉复水后粒径分布见附图1。对比例1-1～1-28与模型样品相比，粒径增长约60-320nm，提示核酸-脂质纳米颗粒的纳米结构已经受损。可见以蔗糖和海藻糖复配作为冻干保护剂，具有超过两种保护剂单独使用的保护效果，以蔗糖、海藻糖和甘露醇组合效果最佳。由于冷冻干燥过程存在多种应力损伤，如低温应力、冻结应力和干燥应力.其中冻结应力又可分为枝状冰晶的形成、离子浓度的增加、pH值的改变和相分离四种情况。蔗糖、海藻糖和甘露醇以适宜比例混合，以适宜添加量加入mRNA-LNP进行冷冻干燥，三者之间因分子结构特征以范德华吸引力为主，形成更为致密的包裹层，将mRNA-LNP完全包裹，在整个体系里构成完整的“姜形”包裹支架：一方面，粉饼刚性较强，不易崩解，能够对抗多种应力损伤，稳定性好；另一方面，孔隙率高，有益于冻干过程中水分快速蒸发，在复水过程中又因与水分子润湿的比表面积大，能够达到快速复水的效果。虽然实施例1-1与对比例1-29保护剂相同，但对比例1-29与脂质体样品对比，粒径增长超过75nm，说明该保护剂体系具有核酸-LNP专属性，即适合核酸-LNP制剂，而不适于核酸-Lip。
实施例2 扫描电镜(SEM)形态观察
扫描电镜(SEM)操作步骤：利用针头取实施例1-1；1-13；1-14；1-15；1-16；1-17；1-18；1-19；1-20；1-21和对比例1-1少许冻干粉末样品置于导电胶上，然后采用SEM扫描成像，结果参见附图2-12。
可见实施例为“姜形”包裹支架微观结构，而对比例中LNP与保护剂之间未能形成支架结构，保护剂仅能起到分散填充，降低应力损伤的作用。
实施例3 透射电镜(TEM)形态观察
将模型样品和实施例1-1，实施例1-4，实施例1-8，实施例1-12，对比例1-10，加入适量的无核酸酶水稀释后，在铜网上滴加稀释样品，以滤纸从铜网边缘吸去多余样品，再滴加2％磷钨酸染色5s，将多余的磷钨酸以滤纸从铜网边缘吸去，利用透射电子显微镜扫描成像，结果见附图13-18，可见实施例较对比例与模型样品具有相近的粒径分布和微观形态。
实施例4 冻干曲线实验
取实施例1-8的样品500μl至3ml西林瓶中，进行冷冻干燥。冷冻干燥程序：预冻阶段：-60℃4小时；解吸干燥阶段：30℃，2小时，全程真空压力控制≤10Pa；升华干燥阶段：按照下述表3程序进行，得实施例2-1～6和对比例2-1～6。
表3升华干燥程序
mRNA含量、包封率、完整性测定及稳定性表征
稳定性放置条件：2-8℃,1个月。分别测定模型样品，实施例2-1～6，对比例2-1～6，在0点和1个月时，各组mRNA总量、游离mRNA浓度、包封率和mRNA完整性，结果见表4。 以RiboGreen RNA试剂与游离mRNA结合荧光显色，利用荧光分析仪测定游离mRNA量；同时，以Triton X-100破膜，测得mRNA总量，则：
包封率(EE％)＝(mRNA总量-mRNA游离量)/mRNA总量*100％
mRNA完整性测定：先将mRNA-LNP样品稀释后70℃变性，制备Dye-Gel胶，注入压胶器中，接着，分别加入mRNA marker，ladder和sample。利用Agilent 2100生物分析系统以凝胶电泳原理高效测定样品mRNA完整性谱图。模型样品、实施例2-1和实施例2-5在0点的mRNA完整性测定图谱如附图19(A/B/C)所示。由上述实验结果可知，冷冻干燥程序中温和多台阶的升温方式更有利于保护核酸-脂质纳米颗粒的纳米结构，提高mRNA包封率和完整性，并提高冻干制剂的稳定性。
表4 mRNA浓度、包封率及完整性

实施例5 细胞转染效果——Western blot实验
以HEK293细胞作为体外蛋白表达模型细胞。以1×106cells/mL细胞密度于6孔板中铺板，并培养24h，接着，将实施例1-1，实施例1-4，实施例1-8和实施例1-12分别以无核酸酶水复溶，再取模型样品，依次加入孔中，加入量为20μL，混匀后转染4h，换液(含血清)。于培养箱中培养24h后采用PBS消化细胞，并将其转移到离心管后离心(2500r/min,5min)，将细胞收集，加入1mL PBS吹匀，转移到1.5mL的EP管中，离心后取下层细胞。每个样品加入200μL的蛋白裂解液(每100μL CHAPS加入1μL Protease inhibiter)，于冰上裂解1h。置于4℃的离心机中离心(13500r/min,20min)后取上清，将样品进行western blot实验测定蛋白表达量。
首先将电泳装置检漏(水检漏，加满，5min内液面不降，则代表装置可以用)，检漏完成，清干净水分；将置好的12％分离胶(体积分数)加入到电泳装置中，再加入无水乙醇(用于压气泡)，将其静置30min后，分离胶凝固后，将无水乙醇倒掉，采用滤纸吸掉多余的液体；加入浓缩胶并迅速插入梳子，放置30min后，浓缩胶凝固；将此装置放到电泳槽中，加入电泳液(1×SDS-PAGE缓冲液)没过铂丝，拔出梳子，分别依次加入实施例1-1,实施例1-4，实施例1-8和实施例1-12，模型样品的蛋白表达样品；电泳(100V，300mA)，在电泳的过程中观察溴酚蓝以及marker的位置，待其到达凝胶底部后停止；取出电泳后的凝胶，按照浓缩胶和分离胶的分界线处分开，去掉浓缩胶，保留分离胶，将分离胶和滤纸在1×bloting buffer中浸泡，PVDF膜在无水甲醇中浸泡使其活化；按照3层滤纸、1层PVDF膜、分离胶、3层滤纸的顺序放在转膜仪上并将气泡赶出，打开电源，15V(5V到15V梯 度升压)，300mA，转印1h 40min；将PVDF膜(转有蛋白)浸于含有5％脱脂奶粉的1×PBST中，在室温下封闭2h，2h后使采用1×PBST洗去过量的脱脂奶粉；将膜放入含有一抗(按照说明书采用5％脱脂奶粉稀释)的15mL离心管中，于4℃孵育过夜，1×PBST洗去过量的一抗；将已经封闭完一抗的PVDF膜放入含有二抗(按照说明书采用5％脱脂奶粉稀释)的15mL离心管中，在室温条件下孵育1h(注意避光)，洗去过量的二抗后，采用Odyssey红外激光成像系统检测并分析。
实验结果见附图20，可见实施例1-1，实施例1-4，实施例1-8和实施例1-12复水制剂与模型样品的S蛋白表达量无显著差异，说明各组的细胞转染效率无显著差异，冷冻干燥工艺并未降低mRNA-LNP的细胞水平生物活性。
实施例6体内免疫效应
取BALB/c小鼠，以实施例1-1，实施例1-4，实施例1-8和实施例1-12复水制剂与模型样品作对比研究，将小鼠随机分为5组，分别肌肉注射50μl实施例1-1，实施例1-4，实施例1-8和实施例1-12复水制剂，型样品进行接种免疫：初次免疫后14天，进行等剂量的第二次免疫；在初次免疫后第14天和28天收集血清，检测SARS-CoV-2特异性IgG和中和抗体应答水平。
初次免疫后第14天免疫效应见附图21，初次免疫后第28天免疫效应见附图22，由实验结果可见实施例1-1，实施例1-4，实施例1-8和实施例1-12均能够引起足够的体内免疫应答，在初次免疫后第28天实施例1-8的免疫应答水平甚至超过了模型样品，说明本发明中设计的冷冻干燥工艺能够维持甚至提高mRNA-LNP的体内免疫效应。

Claims (19)

  1. 一种核酸-脂质纳米颗粒制剂，其特征在于，其含有包括以下组分的冷冻干燥保护剂：蔗糖、海藻糖和甘露醇中的一种或多种。
  2. 根据权利要求1所述的核酸-脂质纳米颗粒制剂，其特征在于，所述蔗糖：海藻糖：甘露醇的质量比为5-15：5-11：1-5。
  3. 一种权利要求1所述的核酸-脂质纳米颗粒制剂的制备方法，其特征在于，按照相应配比，将蔗糖、海藻糖、甘露醇溶解在无核酸酶水中。
  4. 一种冷冻干燥保护剂在制备药物中的应用，其特征在于，所述的冷冻干燥保护剂由以下组分组成：蔗糖、海藻糖，所述蔗糖与海藻糖的质量比为5-15：5-11。
  5. 根据权利要求4所述的应用，其特征在于，所述的冷冻干燥保护剂用于核酸-脂质纳米颗粒，优选地，所述核酸-脂质纳米颗粒中包括ssDNA，dsDNA，mRNA，lncRNA，siRNA，saRNA，shRNA，ASO，质粒，circRNA，circDNA，miRNA，CRISPR-Cas，ployI:C，SamRNA或5’-pppRNA。
  6. 冷冻干燥保护剂在制备药物中的应用，其特征在于，所述的冷冻干燥保护剂包括以下组分：蔗糖、海藻糖和甘露醇，所述蔗糖：海藻糖：甘露醇的质量比为5-15：5-11：1-5，所述的冷冻干燥保护剂用于核酸-脂质纳米颗粒，优选地，所述核酸-脂质纳米颗粒中包括ssDNA，dsDNA，mRNA，lncRNA，siRNA，saRNA，shRNA，ASO，质粒，circRNA，circDNA，miRNA，CRISPR-Cas，ployI:C，SamRNA或5’-pppRNA。
  7. 根据权利要求5或6所述的应用，其特征在于，将核酸-脂质纳米颗粒加入到所述冻干保护剂的溶液中进行冷冻干燥。
  8. 根据权利要求7所述的应用，其特征在于，冷冻干燥包括以下步骤：预冻阶段、升华干燥、解吸干燥。
  9. 根据权利要求8所述的应用，其特征在于，所述预冻阶段：温度为-80℃～-50℃，预冻时间为2～6小时。
  10. 根据权利要求8所述的应用，其特征在于，所述升华干燥阶段：温度为-60℃～0℃，终点温度-5℃～0℃，真空压力控制≤10Pa。
  11. 根据权利要求8所述的应用，其特征在于，所述解吸干燥阶段：温度为0℃～35℃，且终点温度为30～35℃，运行时间2～8小时，真空压力控制≤10Pa。
  12. 根据权利要求11所述的应用，其特征在于，升华干燥阶段分2～8段呈梯度升温，每段升温5～30℃，每段维持时间0.5～10小时。
  13. 根据权利要求8所述的应用，其特征在于，所述药物为疫苗。
  14. 根据权利要求13所述的应用，其特征在于，所述的疫苗为用于预防癌症、病毒感染、细菌感染、真菌感染的疫苗。
  15. 根据权利要求14所述的应用，其特征在于，所述的病毒选自：诺如病毒、埃博拉病毒、冠状病毒、巨细胞病毒、登革热病毒、寨卡病毒、肠病毒、肝炎病毒、单纯疱疹病毒、人乳头瘤病毒、流感病毒、马尔堡病毒、麻疹病毒、脊髓灰质炎病毒、狂犬病病毒、轮状病毒。
  16. 根据权利要求14所述的应用，其特征在于，所述的病毒为柯萨奇病毒。
  17. 根据权利要求13所述的应用，其特征在于，所述冷冻干燥剂在所述疫苗中加入量为5-20w/w％。
  18. 根据权利要求4或5所述的应用，其特征在于，所述的药物为口服制剂、肌肉注射制剂、静脉注射制剂或吸入制剂。
  19. 根据权利要求18所述的应用，其特征在于，所述的吸入制剂为雾化吸入剂或干粉吸入剂。
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