Use of a sugar ester compound or a salt thereof in cryoprotection and freeze-drying protection

Abstract

The present invention provides the use of a sugar ester compound or a salt thereof in cryoprotection and freeze-drying protection, belonging to the field of pharmaceutical technology. The present invention uses the sugar ester compound or a salt thereof as a freeze-drying protectant or a cryoprotectant, which can effectively ensure the integrity of lipid nanoparticles after freeze-drying, the activity of encapsulated drugs, and the particle size of lipid nanoparticles within an acceptable range. Moreover, the appearance of the freeze-dried powder of lipid nanoparticles containing active ingredients is good, and it is easily redissolved (about 5-10 seconds), and the appearance of the sample after redissolution is no different from that before freeze-drying. It can be stored at room temperature for a long time, solving the transportation and storage problems for products stored at low temperature. The composition containing a sugar ester compound or a salt thereof for cryoprotection or freeze-drying protection proposed by the present invention has a wide range of applications. It can not only be used as a freeze-drying / cryoprotectant for lipid nanoparticles, but also as a cryoprotectant for cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to the application of a sugar ester compound or a salt thereof in cryoprotection and freeze-drying protection. Background Art

[0002] Lipid nanoparticles play an important role in improving the drug property and safety of nucleic acid therapy and small molecule drug compound therapy as delivery carriers. For example, in the COVID-19 nucleic acid vaccine, the stability of messenger nucleic acid is effectively improved, and the transfection efficiency of nucleic acid is increased. However, the lipid nanoparticle delivery technology itself still has stability problems, such as easy particle aggregation, oxidation or hydrolysis degradation, which is not conducive to the storage or use of drugs. At present, the two commercially available messenger nucleic acid vaccines: the mRNA vaccine of Moderna (trade name: Spikevax, code: mRNA-1273) needs to be stored and transported at -20°C, and the mRNA vaccine of Pfizer / BioNTech (trade name: Comirnaty, code: BNT162b2) needs to be stored and transported at -70°C, which increases the difficulty and cost of vaccine storage and transportation and is not conducive to the application of the vaccine in more regions. Therefore, the storage stability problem of lipid nanoparticle preparations needs to be solved urgently. A solution for improving the storage capacity of lipid nanoparticle preparations is to manufacture the lipid nanoparticle preparations into freeze-dried products, which can be reconstituted before administration. The freeze-dried lipid nanoparticle composition can be stored at a suitable temperature, which is more convenient for transportation and clinical use.

[0003] Lyophilization is a drying process in which the solvent and / or suspension medium are crystallized at low temperature and then directly sublimated from the solid state to the gaseous state. Lyophilization technology has very prominent advantages, such as maintaining the activity of samples, enhancing the stability and re-solubility of products, simplifying aseptic treatment, accurate dosing, extending the shelf life of preparations, removing water without overheating the product, etc. Lyophilization technology is widely used in pharmaceuticals, biological products and medical products, such as proteins, hormones, viruses, vaccines, bacteria, yeasts, antibiotics, cytostatic agents, liposomes, nanoparticles, antibodies, implants, collagen, etc. However, there are many problems in the lyophilization process. For example, during the lyophilization of lipid nanoparticles, the nanoparticle structure is damaged to varying degrees, resulting in nanoparticle aggregation or precipitation, leading to a significant increase in the particle size of the re-dissolved lipid nanoparticles or drug leakage and inactivation. Conventional lyophilization protectants such as sucrose or trehalose can play a filling role, reduce lyophilization collapse, and improve the appearance of the lyophilized cake, but they cannot protect the microstructure stability of lipid nanoparticles during lyophilization. Some research reports have shown that when mRNA lipid nanoparticles with 10% sucrose as the lyophilization protectant are lyophilized, the particle size after re-dissolution increases significantly and the particle size distribution is poor. Therefore, there is an urgent need to develop better lyophilization solutions for the quality stability problem of lipid nanoparticles.

[0004] Currently, some studies have tried to use sugars, polyols and other water-soluble polymers as lyophilization protectants for lipid nanoparticles. Although there are some improvements in the appearance of lyophilized products, etc., the stability problem of lipid nanoparticles during lyophilization has not been effectively solved.

[0005] Sugars are commonly used lyophilization excipients. During the lyophilization process, oligosaccharides, especially disaccharides, are often used as lyophilization protectants because disaccharides can function as cryoprotectants during the freezing process and as dehydrating protectants during the drying and dehydration process. Sucrose and trehalose are the most commonly used lyophilization protectants in the lyophilization of foods, pharmaceuticals and organisms. Generally, within a certain concentration range, the protective effect of sugars increases with the increase in concentration; when a certain concentration is reached, the protective effect reaches the maximum value. After that, if the sugar concentration is further increased, the protective effect will instead decrease, and with the increase in sugar concentration, the appearance of the lyophilized product becomes worse and worse.

[0006] Tanaka et al. studied the lyophilization protective effects of polyols such as sorbitol, inositol and mannitol on liposomes. The results showed that the lyophilization protective effects of polyols were all worse than those of disaccharides. There were obvious aggregation phenomena in the lyophilized products, the particle size increased significantly after re-dissolution, and the drug leakage was serious.

[0007] It has also been reported that polymers such as polyethylene glycol and polyvinylpyrrolidone, and inorganic salts such as sodium chloride and potassium chloride are added as lyophilization protectants. However, when the degree of polymerization of the polymer is too large, it will crystallize during the freezing process, thus losing the protective effect on the sample. Moreover, when the concentration and molecular weight of the polymer are too large, it will increase the moisture content of the final product, making the sample in the product more unstable; inorganic salts are prone to crystal precipitation during the lyophilization process, which is likely to cause damage to the sample.

[0008] Patent CN114557971A discloses "A Lyophilization Protectant for Nucleic Acid-Lipid Nanoparticles, Its Preparation Method and Application". This patent selects sucrose and trehalose as lyophilization protectants, and the addition amount is 5-20% w / w. Patent CN115624630A discloses "Lyophilization Protecting Composition, Its Application and Method for Cryopreserving Nucleic Acid Lipid Nanoparticles Based on This Composition", which provides a lyophilization protecting composition containing saccharides and reducing agents. The saccharides include a combination of monosaccharides and disaccharides, and the reducing agent is at least one of citric acid, potassium citrate, sodium citrate, ascorbic acid, potassium ascorbate and sodium ascorbate. By mass percentage, the lyophilization protectant contains a PBS solution of 5-20% monosaccharide, 5-20% disaccharide and 0.1-3% reducing agent. This patent also discloses the cryopreserving method of the lipid nanoparticles. Patent CN114727964A discloses "Lyophilization Composition of Lipid Nanoparticles", which discloses the lyophilization technology of blank lipid nanoparticles. The lyophilized lipid nanoparticles are incubated with a nucleic acid solution for drug loading. The weight ratio of the cryoprotectant to the total lipid is 10:1-1000:1, and the concentration of the cryoprotectant is 80-800 mg / ml (calculated before lyophilization). This cryoprotectant is sucrose.

[0009] In summary, after lyophilization and reconstitution, lipid nanoparticle preparations often have problems such as poor particle size, particle size dispersity, encapsulation efficiency and in vivo drug activity of the nanoparticles, and cannot maintain the activity of the original nanoparticles. Currently, the commonly used lyophilization protectants are mainly disaccharide compounds such as sucrose, trehalose, maltose, as well as buffer salts, surfactants, and organic solvents (glycerol). Not only are the types single, but the dosage of sugar is very high, and the lyophilization protection effect is limited. Therefore, there is an urgent need to develop a new type of lyophilization protectant to improve the protection efficiency of the lyophilization protectant and make the product obtained after lyophilization maintain good quality attributes before lyophilization.

[0010] In addition, freezing, as a component step of freeze-drying, also serves as a method for cryopreservation of cells or lipid nanoparticles. Sucrose, trehalose, etc. are also commonly used cryoprotectants for cells or lipid nanoparticles. During the low-temperature freezing process, there is also lipid membrane damage similar to that in freeze-drying, causing membrane fusion or inhibiting enzyme activity. Therefore, in the field of low-temperature freezing technology, it is also necessary to develop safer and more effective cryoprotectants to meet the requirements of low-temperature freezing. Summary of the Invention

[0011] In view of the problems existing in the prior art, the present invention provides the application of a sugar ester compound or its salt in cryoprotection and freeze-drying protection. The present invention uses the sugar ester compound or its salt as a freeze-drying protectant or a cryoprotectant, which can effectively ensure the integrity of lipid nanoparticles after freeze-drying, the activity of encapsulated drugs, and the particle size of lipid nanoparticles within an acceptable range. Moreover, the freeze-dried powder of lipid nanoparticles containing active ingredients has a good appearance after freeze-drying, is easily redissolved (about 5 - 10 seconds), and the appearance of the sample after redissolution is no different from that before freeze-drying, and it can be stored for a long time at room temperature, solving the transportation and storage problems for products stored at low temperatures. The composition containing a sugar ester compound or its salt for cryoprotection or freeze-drying protection proposed by the present invention has a wide range of applications. It can not only be used as a freeze-drying / cryoprotectant for lipid nanoparticles, but also as a cryo / cryopreservation protectant for cells.

[0012] To achieve the above object, in the first aspect, the present invention provides the application of a sugar ester compound or its salt in cryoprotection and freeze-drying protection; the sugar ester compound is obtained by esterifying a sugar with an acid; the sugar is selected from monosaccharides and / or oligosaccharides; the oligosaccharide is composed of 2 - 10 sugar units; the acid is an organic acid and / or an inorganic acid.

[0013] The inventors of the present invention found during the research process that the sugar ester compound or its salt described in the present invention is an amphiphilic molecule, which plays a superimposed protective role on nanoparticles during freezing and / or freeze-drying through molecular-molecular interactions with the filler.

[0014] The salt is obtained by modifying the parent compound by converting an existing acidic or basic moiety into its salt form (e.g., by reacting the free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic salts of basic residues such as amines; alkali metal salts or organic salts of acidic residues such as carboxylic acids. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, tosylate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. The pharmaceutically acceptable salts of the present invention include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids.

[0015] In a preferred embodiment, the acid is selected from sulfuric acid, phosphoric acid, sulfonic acid, methanesulfonic acid, carboxylic acid.

[0016] In a preferred embodiment, the monosaccharide or the sugar units of the oligosaccharide have 4-7 carbon atoms; more preferably furanose and / or pyranose.

[0017] In a preferred embodiment, the monosaccharide or the sugar units of the oligosaccharide are at least one of arabinose, xylose, lyxose, ribose, glucose, mannose, allose, galactose, fructose, sorbose, fucose.

[0018] In a preferred embodiment, the oligosaccharide is selected from oligosaccharides composed of 2-8 sugar units.

[0019] In a preferred embodiment, the oligosaccharides include at least one of disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, and low-molecular-weight glycosaminoglycans; the disaccharides are selected from sucrose, lactose, maltose, trehalose, lactulose, cellobiose, aspergillus sugar, kojibiose, sakebiose, isomaltose, sophorose, laminaribiose, gentiobiose, turanose, isomaltulose, gentiodisulose, mannotriose, melibiose, and xylobiose; the trisaccharides are selected from raffinose and maltotriose; the tetrasaccharide is stachyose; the pentasaccharides are selected from guluronic acid pentasaccharide and pentosan; the hexasaccharide is α-cyclodextrin; the heptasaccharide is β-cyclodextrin; the octasaccharide is γ-cyclodextrin; the low-molecular-weight glycosaminoglycans are selected from at least one of hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin.

[0020] In a preferred embodiment, the sugar ester compound or its salt includes at least one of sodium / potassium fructose diphosphate / sulfate, potassium / sodium glucose diphosphate / sulfate, potassium / sodium glucose diphosphate / sulfate tetra(cyclohexylammonium salt), potassium / sodium glucose pentasulfate / phosphate, sodium / potassium glucose-6-phosphate disodium / potassium salt, trisodium / potassium 6-phosphogluconate, bis(cyclohexylammonium) fucose 1-phosphate / sulfate, bis(cyclohexylammonium) ribose phosphate / sulfate, sodium / potassium galactose sulfate / phosphate, sodium / potassium galactose pentaacetate, galacturonic acid, sodium galacturonate, potassium galacturonate, sodium / potassium mannose phosphate, sodium / potassium pentaacetylmannose ester, disodium / potassium pyranomannose diphosphate ester, disodium / potassium arabinose-5-phosphate, potassium / sodium sucrose heptasulfate, potassium / sodium sucrose hexasulfate, triethylamine salt of sucrose octasulfate, potassium / sodium sucrose octasulfate, sucrose 6,6'-diphosphate, dipotassium salt of sucrose 6'-monophosphate, potassium / sodium lactobionate, lacturonic acid, β-maltose heptaacetate, maltobionic acid, sodium maltobionate, potassium maltobionate, disodium / potassium trehalose-6-phosphate, potassium alginate, sodium heparin disaccharide IS, sodium chondroitin disaccharide ΔDI-6S, sodium dextran sulfate, sodium pentosan polysulfate, sodium cyclodextrin sulfate, sodium β-cyclodextrin phosphate, sodium carboxymethyl-β-cyclodextrin phosphate, sodium N-acetyl-O-sulfated heparin (heparin IV-A), hyaluronic acid, sodium hyaluronate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate.

[0021] In a second aspect, the present invention provides a composition for cryoprotection or freeze-drying protection, the composition comprising:

[0022] The aforementioned sugar ester compound or its salt, and a filler.

[0023] In a preferred embodiment, the mass ratio of the sugar ester compound or its salt to the filler is 1:500 - 3:1, more preferably 1:400 - 2:1.

[0024] In a preferred embodiment, the filler is a pharmaceutically acceptable excipient.

[0025] In a preferred embodiment, the pharmaceutically acceptable excipient is selected from at least one of sugars, polyols, polymers, salts, and amino acids.

[0026] In a preferred embodiment, the composition for cryoprotection or lyophilization protection is formulated with a liquid medium; the liquid medium is selected from water and / or a buffered salt system with a pH of 6.0 - 8.5.

[0027] In a third aspect, the present invention provides the application of the above composition for cryoprotection or lyophilization protection as a cryoprotectant for lipid particles, a lyophilization protectant, or a cryoprotectant for cells.

[0028] In a fourth aspect, the present invention provides a lyophilization protectant or a cryoprotectant, comprising the above composition for cryoprotection or lyophilization protection.

[0029] In a fifth aspect, the present invention provides a lipid nanoparticle composition containing a cryoprotectant or a lyophilization protectant, including the above cryoprotectant or lyophilization protectant, and lipid nanoparticles; the mass ratio of the cryoprotectant or lyophilization protectant to the lipid nanoparticles is 2:1 - 2000:1.

[0030] In a preferred embodiment, in the lipid nanoparticle composition containing a cryoprotectant or a lyophilization protectant before freezing or lyophilization, the concentration of the cryoprotectant or lyophilization protectant is 50 - 400 mg / ml, more preferably 80 - 350 mg / ml.

[0031] The present invention adds a lyophilization protectant to the pretreated lipid nanoparticle liquid and uses it in combination with lyophilization parameters to obtain a high-quality lyophilized lipid nanoparticle product. The lyophilized product is easy to redissolve and easy to administer as a pharmaceutical formulation.

[0032] As used in the present invention, the term "lyophilization protectant" refers to a substance, compound, or excipient added to a composition to protect active ingredients during the drying stage of lyophilization, help preserve or stabilize the lyophilized product, and / or help make the lyophilized product easier to reconstitute. As used in the present invention, a "cryoprotectant" refers to a substance, compound, or excipient added to a biological or pharmaceutical composition to protect it from freeze damage.

[0033] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues within the scope of reasonable medical judgment without excessive use, with toxicity, irritation, allergic reactions, or other problems or complications being commensurate with a reasonable benefit / risk ratio.

[0034] As used in the present invention, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described in the present invention (e.g., excipients capable of suspending or dissolving the active compound) and having substantially non-toxic and non-inflammatory properties in a drug.

[0035] By using the composition for cryoprotection or lyophilization protection, it can be stably stored at a higher temperature than the lipid nanoparticle liquid. Generally, the lipid nanoparticle liquid is stored at -70°C, which is not a suitable temperature for transportation and storage for facilities lacking equipment capable of reaching and maintaining this temperature. The lyophilized composition can be stably stored at a temperature above -20°C.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The present invention proposes a new use of a sugar ester compound or its salt, namely its application in lyophilization and / or freezing.

[0038] 2. The present invention first uses a sugar ester compound or its salt as a component of a cryoprotectant or lyophilization protectant, and provides a new composition for cryoprotection or lyophilization protection for freezing or lyophilizing lipid nanoparticles and / or cryopreserving cells.

[0039] 3. The composition for cryoprotection or lyophilization protection containing a sugar ester compound or its salt proposed by the present invention can well ensure the structural stability of lipid nanoparticles during freezing or lyophilization;

[0040] 4. Using the composition for cryoprotection or lyophilization protection containing a sugar ester compound or its salt proposed by the present invention as a lyophilization protectant, the appearance of the lyophilized powder of lipid nanoparticles containing active ingredients is good after lyophilization, without phenomena such as collapse, shrinkage, cracking, film formation, and spraying;

[0041] 5. Using the composition for cryoprotection or lyophilization protection containing a sugar ester compound or its salt proposed by the present invention as a lyophilization protectant, lipid nanoparticles containing active ingredients are relatively easy to redissolve (about 5 - 10 seconds), and the appearance of the sample after redissolution is no different from that before lyophilization;

[0042] 6. Using the composition containing a sugar ester compound or a salt thereof for cryoprotection or freeze-drying protection proposed by the present invention as a freeze-drying protectant, before freeze-drying and after reconstitution of the freeze-dried lipid nanoparticles containing the active ingredient, pharmaceutical indices such as particle size, zeta potential, encapsulation efficiency, etc. are consistent with those before freeze-drying, and the particle size distribution of the lipid nanoparticles after reconstitution of the freeze-dried product is good.

[0043] 7. Using the composition containing a sugar ester compound or a salt thereof for cryoprotection or freeze-drying protection proposed by the present invention as a freeze-drying protectant or cryoprotectant, the lipid nanoparticles containing the active ingredient show good activity in in vivo / in vitro tests before freeze-drying and after reconstitution of the freeze-dried product or before freezing and after freeze-thawing.

[0044] 8. Using the composition containing a sugar ester compound or a salt thereof for cryoprotection or freeze-drying protection proposed by the present invention as a freeze-drying protectant, the lipid nanoparticles containing the active ingredient can be stored at room temperature for a long time, solving the transportation and storage problems for products stored at low temperatures.

[0045] 9. The composition containing a sugar ester compound or a salt thereof for cryoprotection or freeze-drying protection proposed by the present invention has a wide range of applications. It can not only be used as a freeze-drying / cryopreservation protectant for lipid nanoparticles, but also as a freeze-drying / cryopreservation protectant for cells. Description of the Drawings

[0046] Figure 1 Pictures of the freeze-dried sample and the reconstituted sample of Example 19. Left: Photo before freeze-drying; Middle: Photo after freeze-drying; Right: Photo after reconstitution of the freeze-dried product;

[0047] Figure 2 Particle size distribution diagram of the sample before freeze-drying / after reconstitution of the freeze-dried product of Example 9;

[0048] Figure 3 Particle size distribution diagram of the sample before freeze-drying / after reconstitution of the freeze-dried product of Example 19;

[0049] Figure 4 Scanning electron microscope photo of the freeze-dried sample of Example 15;

[0050] Figure 5 Scanning electron microscope photo of the freeze-dried sample of Example 18;

[0051] Figure 6 Scanning electron microscope photo of the freeze-dried sample of Example 20;

[0052] Figure 7 In vitro cell transfection effect diagram of the sample before freezing / after freeze-thawing of Example 9;

[0053] Figure 8In vitro cell transfection effect diagrams of the samples before freeze-drying / after reconstitution by freeze-drying in Example 11;

[0054] Figure 9 In Example 26 and 30, the curve graph of the change in the tumor volume of mice over time after administration of the samples before freeze-drying / reconstituted by freeze-drying. Detailed implementation manners

[0055] The present invention will be further elaborated in detail below in combination with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments are usually in accordance with conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0056] The technical solutions and beneficial effects of the present invention will be further described below in combination with the embodiments.

[0057] Examples and comparative examples Lipid nanoparticle compositions containing cryoprotectants

[0058] Lipid particles: Luciferase mRNA (Luc mRNA) was prepared into lipid nanoparticles as model sample 1; Docetaxel was prepared into liposomes as model sample 2.

[0059] Cryoprotectant: Weigh the raw materials according to the formula and dissolve them with sterile water to obtain.

[0060] (1) Preparation of lipid nanoparticle compositions containing cryoprotectant / lyoprotectant based on model sample 1:

[0061] Dilute luciferase mRNA in 10 - 100 mM sodium citrate buffer solution with pH 4.0, and the concentration of the mRNA solution is 135 μg / ml; Prepare a lipid mixed ethanol solution according to the molar ratio of ALC-0315:DSPC:cholesterol:ALC-0159 of 47.3:9.6:41.4:1.7 (ALC-0315 is the cationic lipid of the commercial product Comirnaty); After mixing the mRNA solution and the lipid mixed solution in a volume ratio of 3:1 in a nano-drug preparation device, filter through ultrafiltration, collect the sample, and store it at 4°C.

[0062] Prepare lipid nanoparticle compositions containing lyoprotectants according to the prescription compositions and dosages listed in Examples 1 - 23 and Comparative Examples 1 - 6 in Table 1. After adding the lyoprotectant to the model sample and mixing evenly, aliquot it into vials and freeze-dry. The samples after freeze-drying are reconstituted with sterile water.

[0063] Prepare the lipid nanoparticle composition containing cryoprotectant according to the prescription composition and dosage listed in Examples 1-10 of Table 1. After adding the protectant to the model sample and mixing evenly, aliquot it into vials and store it frozen at -80°C. Take out the sample after one month and thaw it at 2-8°C.

[0064] (2)Preparation of the lipid nanoparticle composition containing cryoprotectant / freeze-drying protectant based on Model Sample 2:

[0065] Using DSPC:cholesterol molar ratio of 2:1 as the lipid film material and drug-lipid mass ratio of 1:15, prepare docetaxel liposomes by the thin film dispersion method and collect the samples.

[0066] Prepare the lipid nanoparticle composition containing freeze-drying protectant according to the prescription composition and dosage listed in Examples 24-33 of Table 1. After adding the freeze-drying protectant to the model sample and mixing evenly, aliquot it into vials and freeze-dry. Reconstitute the freeze-dried sample with sterile water.

[0067] Table 1

[0068]

[0069]

[0070] Application Examples

[0071] 1. Quality attributes and stability (particle size, PDI, zeta potential, and encapsulation efficiency) of the freeze-dried and reconstituted samples, as well as the quality attributes of the frozen samples after thawing.

[0072] 1) Particle size and PDI: Measure the average particle size and polydispersity index PDI of the sample solutions in the examples by dynamic light scattering using a Malvern ZetaSizer Nano ZS90. The measurement angle is 90°, the refractive index of the dispersant is 1.330, and the test temperature is 25°C.

[0073] 2) Zeta potential: Measure the Zeta potential of the sample solutions in the examples using a Malvern ZetaSizer Nano ZS90 based on electrophoretic light scattering (ELS). Refractive index of the dispersant: 1.330, test temperature: 25°C.

[0074] 3) Encapsulation efficiency: According to the manufacturer's instructions, the Quant-it Ribogreen RNA Quantification Kit (ThermoFisher Scientific, UK) was used to measure and calculate the encapsulation efficiency of mRNA in the model sample 1. The HPLC method was used to measure the encapsulation efficiency of the model 2 sample. The specific chromatographic conditions were as follows: acetonitrile: water (50:50) was used as the mobile phase, the flow rate was 1.0 ml / min, the injection volume was 20 μl, and the detection wavelength was 232 nm.

[0075] The particle size, PDI, zeta potential and encapsulation efficiency results of the lyophilized and reconstituted lipid nanoparticle compositions containing cryoprotectants / cryodesiccants of the model samples 1-2, as well as those of the examples and comparative examples, are shown in Table 2. The particle size, PDI, zeta potential and encapsulation efficiency results of the cryoprotectant compositions of Examples 1-10 after freeze-thawing are shown in Table 3. The storage stability of the lyophilized samples is shown in Table 4. The pictures of the lyophilized samples and reconstituted samples of Example 19 (left: photo before lyophilization; middle: photo after lyophilization; right: photo after reconstitution of the lyophilized product) are shown in the appendix Figure 1 , and the particle size distribution diagrams of the samples before lyophilization / after reconstitution of the lyophilized product of Example 9 and Example 19 are shown in the appendix Figure 2-3 .

[0076] Table 2 Quality attribute data of the lyophilized and reconstituted samples

[0077]

[0078]

[0079] Table 3 Quality attribute data of the freeze-thawed samples

[0080]

[0081] Table 4 Storage stability data of the lyophilized samples

[0082]

[0083] It can be seen that the key parameters such as particle size, PDI, zeta potential and encapsulation efficiency of the samples in the examples changed very little before and after lyophilization and reconstitution, and there was no significant change compared with the key quality parameters of the model samples. However, there were significant differences in the key quality parameters between Comparative Examples 1-6 and the model samples after lyophilization and reconstitution (Tables 2-3).

[0084] In particular, as can be seen from Table 4, the lipid nanoparticle compositions containing cryoprotectants / cryodrying protectants of Example 1, Example 3, Example 9, Example 12, Example 19, Example 23, Example 26 and Example 30 were redissolved after being stored for 12 months. Compared with direct redissolution without storage, the key parameters such as particle size, PDI, zeta potential and encapsulation efficiency changed very little, and there were no significant changes in the key quality parameters compared with the model sample. For Comparative Example 1, Comparative Example 4 and Comparative Example 6, after being stored for 1 month and then redissolved, compared with direct redissolution without storage, the key parameters such as particle size and PDI changed to a certain extent, and there were significant differences in the key quality parameters compared with the model sample. Thus, it can be seen that the protectants within the scope defined by the present invention can ensure that the key quality parameters of the redissolved samples after freeze-drying are not significantly different from those of the model sample and there are no significant changes even after long-term storage.

[0085] 2. Observation of Scanning Electron Microscope Morphology

[0086] Take the lyophilized powders of Example 15, Example 18 and Example 20, and observe the morphology of the lyophilized samples by a scanning electron microscope magnified 200 times. The results are shown in the appendix Figure 4-6 . It can be seen that the lyophilized samples of Example 15 / 18 / 20 are uniform and loose powders, without stratification or collapse.

[0087] 3. Cell Experiments

[0088] The in vitro transfection efficiency of the lipid nanoparticle composition of Example 9 before and after freezing and thawing and the lipid nanoparticle composition of Example 11 before and after lyophilization was evaluated using HEK-293 cells. The HEK293 cells were routinely cultured in DMEM + 10% FBS medium to ensure that the cells were in the logarithmic growth phase; one day before transfection, they were seeded on a 96-well culture plate at an appropriate cell density and grown overnight. At the time of transfection, the cells should reach 70-90% confluence; the lipid nanoparticle composition of Example 9 before and after freezing and thawing and the lipid nanoparticle composition of Example 11 before lyophilization and after redissolution were diluted into 4 different dose concentrations with DMEM respectively and added to the 96-well cell culture plate so that the concentration in each well reached 400 ng, 200 ng, 100 ng and 50 ng respectively. Lipofectamine 2000 transfected with Luciferase plasmid was used as the positive control (PC). After incubation in a 37 °C, 5% CO2 incubator for 24 h, the substrate was added to the wells, and the fluorescence intensity was measured with an enzyme-labeled instrument. The results are shown in the appendix Figure 7-8 . It can be seen that the samples of Example 9 before and after freezing and thawing and the samples of Example 11 before lyophilization and after lyophilization and redissolution have comparable in vitro transfection effects.

[0089] 4. Animal Experiments

[0090] CB17 Scid female mice at 6 - 8 weeks of age were used. Human glioblastoma cell line (U87) was inoculated subcutaneously into the mice, and the cell seeding amount was 5*10 6 cells / 0.2 mL DPBS with matrigel(1:1) to establish a mouse U87 tumor model.

[0091] For the samples before freeze - drying and the reconstituted samples after freeze - drying in Example 26 and Example 30, they were administered by intravenous injection at a dose of 10 mg / kg; the changes in tumor volume over time after administration were recorded to evaluate the anti - tumor activity of the samples. The results are shown in the appendix Figure 9 . It can be seen that the samples before freeze - drying and the reconstituted samples after freeze - drying in Example 26 and Example 30 have comparable inhibitory effects on U87 tumors and have good anti - tumor activity before and after freeze - drying.

[0092] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a composition for cryoprotection or freeze-drying protection in the preparation of a cryoprotectant for lipid particles or a freeze-drying protectant for lipid particles, characterized in that: The composition of the composition is a sugar ester compound or a salt thereof, and a filler; the mass ratio of the sugar ester compound or a salt thereof to the filler is 1:500 - 3:1; the filler is a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient is selected from at least one of trehalose, pentosan, cellobiose, fructan, cyclodextrin, dextran, sucrose, mannotriose, xylobiose, arabinose, galactose, mannose, lactulose, maltotriose, sorbose, glucose, mannitol, xylitol, polyethylene glycol, sodium carboxymethyl cellulose, sodium carboxymethyl starch, polysorbate, PVP K15, polyethyleneimine, gelatin, hydroxypropyl cellulose, arabic gum, poloxamer, pectin, aspartic acid; The sugar ester compound or a salt thereof is selected from at least one of sodium fructose diphosphate, sodium fructose disulfate, potassium fructose diphosphate, potassium fructose disulfate, potassium glucose diphosphate, sodium glucose diphosphate, potassium glucose disulfate, sodium glucose disulfate, potassium salt of glucose pentasulfate, sodium salt of glucose pentasulfate, potassium salt of glucose pentaphosphate, sodium glucose pentaphosphate, tripotassium salt of 6-phosphogluconic acid, trisodium salt of 6-phosphogluconic acid, fucose 1-phosphate bis(cyclohexylammonium) salt, fucose 1-sulfate bis(cyclohexylammonium) salt, ribose phosphate bis(cyclohexylammonium) salt, ribose sulfate bis(cyclohexylammonium) salt, sodium salt of galactose sulfate, potassium salt of galactose sulfate, sodium salt of galactose phosphate, potassium salt of galactose phosphate, sodium mannan phosphate, potassium mannan phosphate, disodium diphosphorylated mannan pyranoside, dipotassium diphosphorylated mannan pyranoside, disodium arabinose-5-phosphate, dipotassium arabinose-5-phosphate, potassium sucrose heptasulfate, sodium sucrose heptasulfate, potassium sucrose hexasulfate, sodium sucrose hexasulfate, triethylamine salt of sucrose octasulfate, potassium sucrose octasulfate, sodium sucrose octasulfate, sucrose 6,6'-diphosphate, dipotassium sucrose 6'-monophosphate, dipotassium trehalose-6-phosphate, disodium trehalose-6-phosphate, heparin disaccharide IS, sodium salt, chondroitin disaccharide ΔDI-6S sodium salt, sodium pentosan sulfate, sodium cyclodextrin sulfate, sodium β-cyclodextrin phosphate; In the application, the mass ratio of the cryoprotectant for lipid particles or the lyoprotectant for lipid particles in freeze-drying to the lipid nanoparticles is 2:1 - 2000:

1.

2. The application according to claim 1, characterized in that: The sugar ester compound or its salt is selected from at least one of sodium 1,6-diphosphate fructose, Alpha-D-glucose 1,6-diphosphate potassium salt hydrate, potassium glucosepentasulfate, trisodium 6-phosphogluconate, β-L-fucose 1-phosphate bis(cyclohexylammonium) salt, D-ribose 1-phosphate bis(cyclohexylammonium) salt, D-galactose-6-O-sulfate sodium salt, sodium mannan phosphate, guanosine 5'-(trihydrogen diphosphate) P'-D-mannopyranoside disodium salt, D-arabinose-5-phosphate disodium salt, sucrose heptasulfate potassium, sucrose hexasulfate potassium salt, triethylammonium sucrose octasulfate, potassium sucrose octasulfate, sodium sucrose octasulfate, sucrose 6,6'-dicarboxylic acid, dipotassium sucrose 6'-monophosphate, dipotassium trehalose 6-phosphate, heparin disaccharide IS, sodium salt, chondroitin disaccharide ΔDI-6S sodium salt, sodium pentosan polysulfate, sodium cyclodextrin sulfate, sodium β-cyclodextrin phosphate.

3. Use of a composition for cryoprotection or freeze-drying protection in the preparation of a cryoprotectant for lipid particles or a freeze-drying protectant for lipid particles, characterized in that: The composition of the composition is a sugar ester compound or its salt, and a filler; the mass ratio of the sugar ester compound or its salt to the filler is 1:500 - 3:1; the filler is a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient is selected from at least one of trehalose, pentosan, cellobiose, fructan, cyclodextrin, dextran, sucrose, mannotriose, xylobiose, arabinose, galactose, mannose, lactulose, maltotriose, sorbose, glucose, mannitol, xylitol, polyethylene glycol, sodium carboxymethylcellulose, sodium carboxymethyl starch, polysorbate, PVP K15, polyethyleneimine, gelatin, hydroxypropyl cellulose, arabic gum, poloxamer, pectin, aspartic acid. The sugar ester compound is obtained by esterifying a sugar with an organic acid, and the sugar ester compound or its salt is at least one of β-maltose heptaacetate and sodium carboxymethyl-β-cyclodextrin. In the application, the mass ratio of the cryoprotectant for lipid particles or the lyoprotectant for freeze-drying lipid particles to the lipid nanoparticles is 2:1 - 2000:

1.

4. Use of a composition for cryoprotection or lyophilization protection in the preparation of a cryoprotectant for lipid particles or a lyophilization protectant for lipid particles, characterized in that: The composition of the composition is a sugar acid compound or its salt, and a filler; the mass ratio of the sugar acid compound or its salt to the filler is 1:500 - 3:1; the filler is a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient is selected from at least one of trehalose, pentosan, cellobiose, fructan, cyclodextrin, dextran, sucrose, mannotriose, xylobiose, arabinose, galactose, mannose, lactulose, maltotriose, sorbose, glucose, cyclodextrin, mannitol, xylitol, polyethylene glycol, sodium carboxymethylcellulose, sodium carboxymethyl starch, polysorbate, PVP K15, polyethyleneimine, gelatin, hydroxypropyl cellulose, arabic gum, poloxamer, pectin, aspartic acid. The sugar acid compound or its salt is selected from at least one of galacturonic acid, potassium lactose, sodium lactose, lactobionic acid, maltobionic acid, potassium alginate. In the application, the mass ratio of the cryoprotectant for lipid particles or the lyoprotectant for freeze-drying lipid particles to the lipid nanoparticles is 2:1 - 2000:

1.

5. Use of a composition for cryoprotection or lyophilization protection in the preparation of a cryoprotectant for lipid particles or a lyophilization protectant for lipid particles, characterized in that: The composition comprises an oligosaccharide or its salt, and a filler; the mass ratio of the oligosaccharide or its salt to the filler is 1:500 - 3:1; the filler is a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient is selected from at least one of trehalose, pentosan, cellobiose, fructan, cyclodextrin, dextran, sucrose, mannotriose, xylobiose, arabinose, galactose, mannose, lactulose, maltotriose, sorbose, glucose, mannitol, xylitol, polyethylene glycol, sodium carboxymethyl cellulose, sodium carboxymethyl starch, polysorbate, PVP K15, polyethyleneimine, gelatin, hydroxypropyl cellulose, arabic gum, poloxamer, pectin, aspartic acid; The oligosaccharide is composed of 2 - 6 sugar units; the oligosaccharide is selected from at least one of hyaluronic acid, chondroitin sulfate and N-acetyl-O-sulfated heparin sodium salt; In the application, the mass ratio of the cryoprotectant for lipid particles or the lyoprotectant for lipid particles to the lipid nanoparticles is 2:1 - 2000:1; The molecular weight of the N-acetyl-O-sulfated heparin sodium salt is 1526.03; the molecular weight of the chondroitin sulfate is 499.37; the molecular weight of the hyaluronic acid is 1200.

6. The application according to any one of claims 1-5, characterized in that: The mass ratio of the sugar ester compound or its salt, the sugar acid compound or its salt, or the oligosaccharide or its salt to the filler is 1:400 - 2:

1.

7. The application according to any one of claims 1-5, characterized in that: The composition for cryoprotection or lyoprotection is formulated with a liquid medium.

8. The application according to claim 7, wherein: The liquid medium is selected from water and / or a buffer salt system with a pH of 6.0 - 8.5.

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