New application of glycoside alkaloid and preparation composition of glycoside alkaloid

Abstract

The invention belongs to the technical field of biological medicine, and provides application of glycoside alkaloid to promotion of mRNA expression and promotion of DCs maturation and activation and a pharmaceutical composition containing the glycoside alkaloid. According to the glycoside alkaloid and the pharmaceutical composition provided by the invention, the immunogenicity of mRNA vaccines is greatly improved, the immune response is activated, and the purpose of preventing and treating diseases is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a use of glycoside alkaloids, and a preparation composition containing the glycoside alkaloids and the use thereof. Technical background:

[0002] As a new generation of treatment options, mRNA vaccines have unique advantages in effectiveness, personalized treatment and rapid production. Its superior timeliness also makes it a focus of attention in the development of new coronavirus vaccines. The working principle of mRNA vaccines is: mRNA encoding specific antigenic proteins is delivered to antigen-presenting cells (mainly dendritic cells / DCs) through a delivery vector, and the host cell expression system synthesizes proteins to provide antigen signals. At the same time, DCs produce auxiliary co-stimulatory signals, thereby inducing the host immune system to produce a specific immune response to the antigen, effectively playing a role in disease treatment and prevention. mRNA vaccines also have great clinical application potential in tumor treatment, and many varieties have entered Phase II clinical studies. Despite this, mRNA vaccines still have problems such as low translation efficiency, limited antigen presentation, and weak immune response, resulting in unsatisfactory efficacy and delayed clinical transformation.

[0003] Glycoalkaloids are important secondary metabolites produced by some plants (especially Solanaceae plants). It is generally believed that their main function is to protect plants from external damage. This type of compound is toxic to humans and animals, but when Solanaceae plants are used as medicines, glycoalkaloids are the active ingredients. Studies have shown that glycoalkaloids have potential anti-tumor, anti-pathogenic microorganism, anti-parasitic and other drug activities. However, there are few studies on the application of glycoalkaloids in the field of mRNA vaccines. Summary of the invention:

[0004] The present invention provides the use of glycoside alkaloids for promoting mRNA expression, enhancing antigen signals, and promoting DCs maturation and activation, as well as a pharmaceutical composition containing glycoside alkaloids. The glycoside alkaloids and pharmaceutical composition provided by the present invention greatly improve the immunogenicity of mRNA vaccines, activate immune response reactions, and achieve the purpose of preventing and treating diseases.

[0005] The present invention provides the use of glycoside compounds in nucleic acid drugs for promoting nucleic acid expression or promoting DCs maturation and activation, wherein the glycoside compounds are selected from one or more of tomatine and its derivatives, tomatine analogs and their derivatives, solanine and its derivatives, solanine and its derivatives, chaconine and its derivatives, and solanine and its derivatives.

[0006] The present invention also provides a preparation composition, comprising a glycoside compound, a nucleic acid drug and a delivery vector; wherein the glycoside compound is selected from one or more of tomatine and its derivatives, tomatine analogs and their derivatives, solanine and its derivatives, solanine and its derivatives, chaconine and its derivatives, and solanine and its derivatives.

[0007] Furthermore, in the preparation composition provided by the present invention, the glycoside compound is tomatidine and its derivatives; preferably, the glycoside compound is tomatidine.

[0008] Furthermore, in the preparation composition, the concentration of the glycoside compound in the preparation composition is 0.3 to 0.8 mM; preferably 0.4 to 0.75 mM;

[0009] Furthermore, in the preparation composition provided by the present invention, the nucleic acid drug is selected from one or more of DNA, ASO, siRNA, miRNA, mRNA, ribozyme, and nucleic acid aptamer.

[0010] Furthermore, in the preparation composition provided by the present invention, the nucleic acid drug is mRNA; wherein the mRNA encodes one or more of viral antigen proteins, protein supplementation therapeutic proteins, tumor antigen proteins, gene editing tool proteins or cytokines.

[0011] Furthermore, in the preparation composition provided by the present invention, the delivery carrier co-carries the glycoside compound and the nucleic acid drug;

[0012] Furthermore, in the preparation composition, the mass (g) ratio of the glycoside compound to the nucleic acid drug is 0.2:1 to 10:1; preferably, the mass (g) ratio of the glycoside compound to the nucleic acid drug is 1:1 to 10:1;

[0013] Furthermore, the mass (g) ratio of the glycoside compound to the nucleic acid drug is preferably 3:1.

[0014] The preparation composition provided by the present invention, wherein the delivery carrier is selected from one or more of lipid nanoparticles, PLGA nanoparticles, micelles, liposomes, core-shell nanoparticles or polymer nanoparticles.

[0015] Furthermore, the preparation composition provided by the present invention, wherein the lipid nanoparticles comprise one or more of cationic lipids, neutral phospholipids, steroids and polyethylene glycol lipids;

[0016] Further, the lipid nanoparticle, wherein the cationic lipid is selected from the following structures of MIC1, MIC2, MIC3, MIC4, MIC5 or MIC6:

[0017]

[0018] The preparation composition provided by the present invention, the lipid nanoparticles, wherein the neutral phospholipids are selected from one or more of 1,2-diol-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylcholine (DMPC), 1,2-bis(dimethylphosphino)ethane (DMPE, dipalmitoylphosphatidylcholine (DPPC), 1,2-bis(diphenylphosphino)ethane (DPPE), diethyl pyrocarbonate (DEPC), hydrogenated soybean lecithin (HSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);

[0019] Furthermore, in the lipid nanoparticles, the molar ratio of the cationic lipid to the neutral phospholipid is 0.1:1 to 20:1; preferably 1:1 to 2:1;

[0020] The preparation composition provided by the present invention, the lipid nanoparticles, wherein the steroid is selected from cholesterol, sitosterol, soysterol, lanosterol, ergosterol or a combination thereof;

[0021] Furthermore, the lipid nanoparticles, wherein the molar ratio of the cationic lipid to the steroid is 1:10 to 10:1; preferably 1:2 to 2:1;

[0022] The formulation composition provided by the present invention, the lipid nanoparticles, wherein the polyethylene glycol lipid is selected from DMG-PEG, DSPE-PEG or a combination thereof;

[0023] Furthermore, in the lipid nanoparticles, the molar ratio of the cationic lipid to the polyethylene glycol lipid is 5:1 to 100:1, preferably 10:1 to 20:1.

[0024] In a specific embodiment of the present invention, the preparation composition comprises tomatidine, mRNA, MIC5, DOPE, cholesterol or DMG-PEG2000;

[0025] Furthermore, the mass ratio (g) of tomatidine to mRNA is 0.5:1-10:1, and the mass ratio of MIC5 to mRNA is 15:1; preferably, the mass ratio of tomatidine to mRNA is 0.5:1-5:1; more preferably, the mass ratio of tomatidine to mRNA is 3:1.

[0026] The preparation composition provided by the present invention, wherein the liposome comprises a cationic lipid and a steroid, wherein the cationic lipid is selected from DODAC (N,N-dioleyl-N,N-dimethylammonium chloride), DDAB (N,N-distearyl-N,N-dimethylammonium bromide), DOTAP (1,2-dioleoyltrimethylammonium propane chloride), DOTMA (N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride), DODMA (N,N-dimethyl-2,3-dioleyloxy)propylamine), DLinDMA (,2-dilinoleyloxy-N,N-dimethylaminopropane), DLenDMA (1,2-dilinoleyloxy-N,N-dimethylaminopropane), γ-DLenDMA (1,2-di-γ-linoleyloxy-N,N-dimethylaminopropane), DLin-C-DAP (,2-dilinoleylcarbamoyloxy- 3-dimethylaminopropane), DLin-DAC (1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane), DLin-MA (1,2-dilinoleyloxy-3-morpholinopropane), DLinDAP (1,2-dilinoleoyl-3-dimethylaminopropane), DLin-S-DMA (1,2-dilinoleylthio-3-dimethylaminopropane), DLin-2-DMAP (1-linoleoyl-2 -linoleyloxy-3-dimethylaminopropane), DOAP (3-(N,N-dioleylamino)-1,2-propylene glycol), DOPE (1,2-dioleoyl-sn-3-phosphoethanolamine), DODAP (1,2-dioleoyl-3-dimethylammonium propane), DMRIE (N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide); preferably DOTAP;

[0027] Further, the liposome, wherein the mass ratio of DOTAP to tomatidine is 0.5:1 to 10:1;

[0028] Furthermore, the liposome, wherein the steroid is selected from one or more of cholesterol, sitosterol, soybean sterol, lanosterol, ergosterol; preferably cholesterol

[0029] Furthermore, in the liposome, the mass ratio of the cationic lipid to cholesterol is 1:1 to 5:1.

[0030] Furthermore, the preparation composition provided by the present invention comprises tomatidine, DOTAP, cholesterol and mRNA; wherein the mass ratio of DOTAP to tomatidine is 0.5:1 to 7:1; preferably 1.2:1 to 1.5:1.

[0031] The present invention also provides use of the preparation composition in preparing mRNA drugs for promoting mRNA expression or promoting DCs maturation and activation.

[0032] The use of the mRNA drug provided by the present invention is for treating infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases, protein deficiency or gene defect diseases; preferably, the disease is selected from infectious diseases and cancer;

[0033] Further, the cancer is selected from nasopharyngeal carcinoma, lung cancer, gastric cancer, lymphoma, and melanoma;

[0034] Furthermore, the infectious disease is selected from the group consisting of coronavirus, influenza virus, diseases caused by HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies or herpes.

[0035] The use provided by the present invention, wherein the preparation composition administers a dosage of about 0.001 mg / kg to about 10 mg / kg of the nucleic acid drug to the mammal; preferably the mammal is a human; further, the dosage of the glycoside compound is 0.2 to 10 times that of the nucleic acid drug; preferably 1 to 10 times; more preferably 3 times.

[0036] In a specific implementation mode of the present invention, the mass (g) ratio of the glycoside compound to the nucleic acid drug in the preparation composition is 1:1 to 10:1;

[0037] In another specific embodiment of the present invention, the mass (g) ratio of the glycoside compound to the nucleic acid drug in the preparation composition is 1:1;

[0038] In another specific embodiment of the present invention, the mass (g) ratio of the glycoside compound to the nucleic acid drug in the preparation composition is 3:1;

[0039] In another specific embodiment of the present invention, the mass (g) ratio of the glycoside compound to the nucleic acid drug in the preparation composition is 5:1;

[0040] In another specific embodiment of the present invention, the mass (g) ratio of the glycoside compound to the nucleic acid drug in the preparation composition is 8:1;

[0041] In another specific embodiment of the present invention, the mass (g) ratio of the glycoside compound to the nucleic acid drug in the preparation composition is 10:1;

[0042] In a specific embodiment of the present invention, the lipid nanoparticles, wherein the molar ratio of the cationic lipid to the neutral phospholipid is 1:1;

[0043] In another specific embodiment of the present invention, the lipid nanoparticles, wherein the molar ratio of the cationic lipid to the neutral phospholipid is 1.5:1;

[0044] In another specific embodiment of the present invention, the lipid nanoparticles, wherein the molar ratio of the cationic lipid to the neutral phospholipid is 2:1;

[0045] In a specific embodiment of the present invention, the lipid nanoparticles, wherein the molar ratio of the cationic lipid to the steroid is 1:2;

[0046] In another specific embodiment of the present invention, the lipid nanoparticles, wherein the molar ratio of the cationic lipid to the steroid is 1:1;

[0047] In another specific embodiment of the present invention, the lipid nanoparticles, wherein the molar ratio of the cationic lipid to the steroid is 2:1;

[0048] In a specific embodiment of the present invention, the lipid nanoparticles, wherein the molar ratio of the cationic lipid to the polyethylene glycol lipid is 10:1;

[0049] In another specific embodiment of the present invention, the lipid nanoparticles, wherein the molar ratio of the cationic lipid to the polyethylene glycol lipid is 15:1;

[0050] In another specific embodiment of the present invention, the lipid nanoparticles, wherein the molar ratio of the cationic lipid to the polyethylene glycol lipid is 20:1;

[0051] In a specific embodiment of the present invention, the liposome, wherein the mass ratio of the cationic lipid to cholesterol is 1:1;

[0052] In another specific embodiment, the liposome, wherein the mass ratio of the cationic lipid to cholesterol is 3:1;

[0053] In another specific embodiment of the present invention, the liposome, wherein the mass ratio of the cationic lipid to cholesterol is 5:1;

[0054] In a specific embodiment of the present invention, the liposome, wherein the mass ratio of DOTAP to tomatidine is 1.2:1;

[0055] In another specific embodiment of the present invention, the liposome, wherein the mass ratio of DOTAP to tomatidine is 1.3:1;

[0056] In another specific embodiment of the present invention, the liposome, wherein the mass ratio of DOTAP to tomatidine is 1.5:1;

[0057] In a specific embodiment of the present invention, the concentration of the glycoside compound in the preparation composition is 0.45 mM;

[0058] In another specific embodiment of the present invention, in the preparation composition, the concentration of the glycoside compound in the preparation composition is 0.75 mM. Description of the drawings:

[0059] Figure 1 . Schematic diagram of the preparation process of the liposome preparation (T-LPX) containing mRNA active ingredients and tomatidine;

[0060] Figure 2 .Particle size, PDI and potential of T-LP containing different concentrations of tomatidine; Among them, Figure 2 A is the particle size and PDI diagram, Figure 2 B is the potential diagram;

[0061] Figure 3 .Particle size and potential diagram of LPX and T-LPX; Figure 3 A is the particle size and PDI diagram, Figure 3 B is the potential diagram;

[0062] Figure 4 .Inverted microscope observation results and flow cytometry detection results of LPX, LPX+T and T-LPX; Among them, Figure 4 A is a microscope observation picture; Figure 4 B is a graph of cytometry detection data;

[0063] Figure 5 .The results of transfection of DC2.4 cells with T-LPX containing different concentrations of tomatidine; Figure 5 A is the transfection result diagram. Figure 5 B is the fluorescence intensity;

[0064] Figure 6 . Cell transfection results of different batches of T-LPX preparations;

[0065] Figure 7 . Cell transfection results of T-LPX stability samples;

[0066] Figure 8 .The results of antigen presentation of LPX and T-LPX containing different concentrations of tomatidine on BMDCs; Figure 8 A is the cell distribution map, Figure 8 B is a bar graph;

[0067] Fig. 9 .The results of LPX and T-LPX containing different concentrations of tomatidine on the maturation and activation of BMDCs; Fig. 9 A is the cell map, Fig. 9 B is a bar graph;

[0068] Fig.10 .Distribution and expression imaging results of LPX and T-LPX loaded with DID and Luc mRNA in mice;

[0069] Fig.11 .Quantitative results of the distribution and expression of LPX and T-LPX in mice;

[0070] Fig.12 . The tumor inhibition results of T-LPX / OVA vaccine preparation loaded with OVA mRNA in E.G7-OVA mouse tumor model; Fig.13 .The results of T-LPX / OVA inducing the production of OVA-specific CD8+T cells; Fig.13 A is the cell map, Fig.13 B is the corresponding bar graph;

[0071] Fig.14 .The results of LPX / OVA and T-LPX / OVA promoting the maturation and activation of DCs in the spleen and lung in vivo; A is the spleen result diagram, and B is the lung result diagram;

[0072] Fig.15 .The results of antigen presentation of LPX / OVA and T-LPX / OVA on DCs in spleen and lymph nodes; A is the result of spleen, and B is the result of lymph nodes;

[0073] Fig.16 . Schematic diagram of the preparation process of lipid nanoparticle preparation (T-NPX) containing mRNA active ingredients and tomatidine;

[0074] Fig.17 .Particle size and potential diagram of NPX and T-NPX; A is the particle size and PDI diagram, and B is the potential diagram;

[0075] Fig.18 .Inverted microscope observation results and flow cytometry detection results of NPX, NPX+T and T-NPX; A is the microscope observation picture; B is the cytometry detection data picture;

[0076] Fig.19 .Results of NPX and T-NPX promoting BMDCs maturation and activation; A is the cell image, and B is the corresponding bar graph;

[0077] Fig. 20. Imaging and quantitative results of the expression of NPX and T-NPX loaded with Luc mRNA in mice; A is the in vivo imaging diagram, and B is the data result diagram;

[0078] Fig.21 . Results of the induction of RBD-specific IgG antibody titers by NPX and T-NPX loaded with Delta variant mRNA at low doses (10 μg) and high doses (30 μg);

[0079] Fig. 22 .Results of NPX and T-NPX inducing T cells in spleen and lymph nodes; A is the imaging image and B is the data bar graph;

[0080] Fig.23 .The results of NPX and T-NPX inducing the production of IFN-γ+T cells; A is the observed image and B is the data bar graph. Specific implementation method:

[0081] The term "cationic lipid" refers to a lipid that has a positive charge at a selected pH value.

[0082] Cationic lipids easily bind to negatively charged nucleic acids, that is, they interact with the negatively charged phosphate groups present in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs).

[0083] In a specific embodiment, the cationic lipid compound of the present invention can be a commercially available cationic lipid, or a cationic lipid compound reported in the literature.

[0084] The therapeutic agent or preventive agent includes one or more of nucleic acid molecules, small molecule compounds, macromolecular compounds, polypeptides, antibodies or proteins.

[0085] The nucleic acid includes but is not limited to single-stranded DNA, double-stranded DNA and RNA. Suitable RNA includes but is not limited to small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

[0086] The delivery vehicle, such as lipid nanoparticles LNP, PLGA nanoparticles, micelles, liposomes, core-shell nanoparticles, polymer nanoparticles, may comprise neutral lipids, such as neutral phospholipids.

[0087] Neutral lipids in the present disclosure refer to lipids that are uncharged or exist in the form of zwitterions at a selected pH value and play an auxiliary role. The neutral lipids may regulate the fluidity of nanoparticles into lipid bilayer structures and improve efficiency by promoting lipid phase transition, and may also affect the specificity of target organs.

[0088] For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols, and derivatives thereof.

[0089] The carrier component comprising the cationic lipid complex may include one or more neutral lipid-phospholipids, such as one or more unsaturated lipids.

[0090] Phospholipids can be assembled into one or more lipid bilayers. In general, phospholipids can include a phospholipid portion and one or more fatty acid portions.

[0091] The neutral lipid part can be selected from the non-limiting group of the following composition: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. The fatty acid part can be selected from the non-limiting group of the following composition: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. Also encompassed are non-natural species including natural species with modification and replacement, the modification and replacement include branching, oxidation, cyclization and alkynes. For example, phospholipids can be functionalized with one or more alkynes (e.g., one or more double bonds are replaced by triple bond alkenyl) or cross-linked with the one or more alkynes. Under appropriate reaction conditions, alkynyl may undergo copper-catalyzed cycloaddition reactions when exposed to azide. These reactions can be used to functionalize the lipid bilayer of the complex to facilitate membrane permeation or cellular recognition, or to couple the complex to useful components such as targeting or imaging moieties (eg, dyes).

[0092] The neutral lipids that can be used in the carrier of the present invention can be selected from the non-limiting group consisting of: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether). PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

[0093] In some embodiments, the cationic lipid is selected from DODAC (N,N-dioleyl-N,N-dimethylammonium chloride), DDAB (N,N-distearyl-N,N-dimethylammonium bromide), DOTAP (1,2-dioleoyltrimethylammonium propane chloride), DOTMA (N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride), DODMA (N,N-dimethyl-2,3-dioleyloxy)propylamine), DLinDMA (,2-dilinoleyloxy-N,N-dimethylaminopropane), DLenDMA (1,2-dilinoleyloxy-N,N-dimethylaminopropane), γ-DLenDMA (1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane), DLin-C-DAP (,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane), DL In-DAC (1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane), DLin-MA (1,2-dilinoleyloxy-3-morpholinopropane), DLinDAP (1,2-dilinoleoyl-3-dimethylaminopropane), DLin-S-DMA (1,2-dilinoleylthio-3-dimethylaminopropane), DLin-2-DMAP (1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane), DOAP (3-(N,N-dioleylamino)-1,2-propylene glycol), DOPE (1,2-dioleoyl-sn-3-phosphoethanolamine), DODAP (1,2-dioleoyl-3-dimethylammonium propane), DMRIE (N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide), preferably DOTAP;

[0094] In some embodiments, the neutral lipid comprises DSPC.

[0095] In some embodiments, the neutral lipid comprises DOPE.

[0096] In some embodiments, the neutral lipids include both DSPC and DOPE.

[0097] The carrier comprising cationic lipids may also include one or more structural lipids, such as steroids. Structural lipids in the present disclosure refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

[0098] The structured lipid can be selected from but not limited to the group consisting of: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, ursolic acid, alpha-tocopherol, corticosteroids and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone and hydrocortisone) or a combination thereof.

[0099] The carrier comprising cationic lipids can also include one or more polymer conjugated lipids, such as PEGylated lipids. Polymer conjugated lipids mainly refer to lipids modified by polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNPs, regulates nanoparticle size by limiting lipid fusion, and increases the half-life of nanoparticles by reducing nonspecific interactions with macrophages.

[0100] In one embodiment, the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. The molecular weight of PEG-modified PEG is generally 350-5000Da.

[0101] For example, the polymer conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) and methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0102] In one embodiment of the present disclosure, the polymer-conjugated lipid is DMG-PEG2000.

[0103] In one embodiment of the present disclosure, the lipid nanoparticles include neutral lipids, structural lipids and polymer-conjugated lipids.

[0104] In certain embodiments, therapeutic and / or preventive agents are mRNA. mRNA can encode any polypeptide of interest, including any natural or non-natural polypeptide or otherwise modified polypeptide. The polypeptide encoded by mRNA can have any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by mRNA can have a therapeutic effect when expressed in a cell.

[0105] In other embodiments, therapeutic and / or preventive agents are siRNA. siRNA can selectively reduce the expression of a gene of interest or down-regulate the expression of the gene. For example, the selection of siRNA can make the gene silencing relevant to a specific disease, disease or condition after the compound comprising the siRNA is administered to a subject in need. siRNA can include a sequence complementary to the mRNA sequence of a gene or protein of interest to encode. In some embodiments, siRNA can be an immunomodulatory siRNA.

[0106] In certain embodiments, the therapeutic and / or preventive agent is sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as a gene editing tool. For example, the sgRNA-cas9 complex can affect the mRNA translation of a cell gene.

[0107] In some embodiments, the therapeutic and / or preventive agent is shRNA or its encoding vector or plasmid. shRNA can be produced inside the target cell after the appropriate construct is delivered to the nucleus. The construct and mechanism associated with shRNA are well-known in the relevant field.

[0108] In some embodiments, the N: P ratio of the formulation composition refers to the molar ratio of the nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. In some embodiments, a lower N: P ratio is selected. One or more RNAs, lipids, and amounts thereof may be selected to provide an N: P ratio of about 2: 1 to about 30: 1, such as 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 22: 1, 24: 1, 26: 1, 28: 1, or 30: 1. In certain embodiments, the N: P ratio may be about 2: 1 to about 8: 1. In other embodiments, the N: P ratio is about 3: 1.

[0109] The formulation composition or carrier disclosed in the present invention (such as plasmid nanoparticles LNP, PLGA nanoparticles, micelles, liposomes, core-shell nanoparticles, polymer nanoparticles, etc.) can deliver active ingredients to subjects or patients, including therapeutic agents or preventive agents. The therapeutic agent or preventive agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins. Therefore, the complex disclosed in the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0110] In a specific embodiment of the present invention, the PLGA nanoparticles are mainly composed of poly(lactic acid-glycolic acid) copolymer nanoparticles.

[0111] In a specific embodiment of the present invention, the liposome is mainly assembled from cholesterol, lipids and their derivatives; the lipid is (1,2-dioleyloxypropyl)trimethylammonium chloride.

[0112] The cationic lipid used in the present invention can be obtained from the existing document Adv. Funct. Mater. 2022, 2204692.

[0113] The structure of MIC1, MIC2, MIC3, MIC4, MIC5 or MIC6 is as follows:

[0114]

[0115] The mRNA used in the present invention can be purchased commercially.

[0116] The specific materials used in the present invention are all commercially available or prepared according to the prior art.

[0117] Example 1 Tomatine enhances the anti-tumor activity of mRNA vaccines based on liposome delivery vector LP

[0118] 1. Preparation of T-LPX

[0119] like Figure 1 As shown, tomatidine-loaded liposomes (T-LP) were prepared by a thin film hydration method, and then electrostatically incubated with mRNA to obtain mRNA liposome complexes (T-LPX), thereby achieving co-loading of tomatidine and mRNA.

[0120] (1) Screening of tomatine content in the preparation of T-LP

[0121] T-LP was prepared by thin film dispersion-hydration ultrasonic method. The specific preparation method is as follows: accurately measure the corresponding prescription amount of tomatidine, DOTAP and Cholesterol stock solution in a 100mL eggplant-shaped bottle, add the corresponding volume of anhydrous ethanol to 3mL, and evaporate at 37℃ under reduced pressure for 2h to remove the anhydrous ethanol to form a uniform lipid film; add 3mL of RNase-free water, and hydrate the lipid film at 60℃ for 40min; the obtained lipid colloid solution is immediately probe ultrasonic for 3min (power 80W), and RNase-free water is added to 3mL to quantify the DOTAP concentration in the preparation to 1.5mM, and filter with a 0.22μm sterile water filter membrane to obtain T-LP. Blank liposome LP was prepared in the same way. Take the corresponding prescription amount of DOTAP and Cholesterol stock solution, add the corresponding volume of anhydrous ethanol, and the remaining steps are exactly the same as the T-LP preparation steps. The recipes for preparing 3 mL of LP and T-LP containing different concentrations of tomatidine (tomatidine concentrations of 0.15 mM, 0.45 mM, 0.75 mM, 1.2 mM, and 1.5 mM) are shown in Table 1 .

[0122] Table 1 Prescription composition of LP and T-LP containing different concentrations of tomatidine

[0123]

[0124] (2) Preparation of T-LP: 4.19 mg of (1,2-dioleyloxypropyl)trimethylammonium chloride (DOTAP), 2.32 mg of cholesterol and 3.1 mg of tomatidine were accurately weighed and dissolved in 3 mL of anhydrous ethanol and added to a 100 mL eggplant-shaped bottle. Then, the anhydrous ethanol was removed by vacuum rotary evaporation at 50 rpm for 2 h in a constant temperature water bath at 37 °C to form a uniform lipid film. Subsequently, 3 mL of RNase-free water was added and the lipid film was hydrated at 60 °C for 40 min; the resulting lipid colloidal solution was immediately subjected to probe ultrasonication for 3 min in an ice bath (power 80 W, stop for 3 s and supersonication for 3 s). Finally, RNase-free water was added to 2 mL to quantitatively adjust the DOTAP concentration in the preparation to 3 mM, and the preparation was filtered using a 0.22 μm sterile water filter to obtain T-LP. The molar ratio of DOTAP to tomatidine in the T-LP obtained with this prescription was 3:1. Blank liposome LP was prepared in the same manner without adding tomatidine.

[0125] (3) Preparation of T-LPX: The T-LP obtained above is electrostatically co-incubated with mRNA to prepare the tomatidine liposome mRNA complex. For example, take 30 μL of T-LP and dilute 10 μg of mRNA with RNase-free water to an equal volume of 30 μL; then slowly add the mRNA dilution to the liposome solution, gently blow and mix, and incubate at room temperature for 10 minutes to obtain T-LPX with N (nitrogen) / P (phosphorus) = 3. The ordinary liposome mRNA complex LPX is prepared in the same way.

[0126] 2. Pharmaceutical investigation of T-LPX

[0127] Take appropriate amount of the above LP, T-LP, LPX and T-LPX formulation solutions and dilute them 10 times with purified water, and use Malvern laser particle size analyzer to measure the formulation properties such as particle size, particle size distribution (PDI) and ζ potential. Each sample is measured three times in parallel, and the measurement temperature is 25°C.

[0128] Depend on Figure 2 It can be seen that Figure 2 In A, the particle size of LP is about 60 nm, and the particle size of T-LP is no higher than 100 nm; the particle size distribution (PDI) is less than 0.3, indicating that the size distribution of LP and T-LP is uniform; Figure 2 In B, the potentials of LP and T-LP are both around 50 mV, indicating that the content of tomatidine has little effect on the potential of the preparation.

[0129] Depend on Figure 3It can be seen that the particle size of LPX and T-LPX is around 150nm ( Figure 3 A), PDI is less than 0.3( Figure 3 A), the potential is about 35mV ( Figure 3 B); This indicates that the addition of tomatidine to co-load mRNA does not affect the particle size and potential of the final liposome mRNA complex.

[0130] 3. In vitro expression of T-LPX

[0131] Using GFP mRNA as a reporter gene and DC2.4 cells, the most commonly used in vitro cell model for nanovaccines, as the research object, the in vitro transfection performance of T-LPX provided by the present invention was evaluated by transfection experiments on DC2.4 cells. The transfection of LPX and T-LPX in DC2.4 cells was investigated by inverted fluorescence microscopy and flow cytometry; in addition, a physical mixture group LPX+T of a common mRNA preparation and tomatidine was set up for experiments.

[0132] The transfection method is as follows:

[0133] (1) Cell plating: DC2.4 cells in the logarithmic growth phase were collected by trypsin digestion, resuspended in DMEM complete medium, and counted. Then, 1×10 5 500 μL / well were inoculated into a 24-well plate, shaken evenly, and cultured overnight in a 37°C, 5% CO2 cell incubator.

[0134] (2) Preparation and administration: The preparation method of the GFP mRNA-loaded tomatidine liposome complex T-LPX for cell transfection experiments is as follows: according to N / P=3 / 1, 6 μL T-LP and 1 μg GFP mRNA were diluted in 50 μL Opti-MEM, and then the mRNA dilution was added to the liposome dilution to mix well and incubate at room temperature for 10 min. The ordinary liposome mRNA complex LPX was prepared in the same way. At the same time, the tomatidine compound solution and the prepared LPX were physically mixed to set up the LPX+T group. Finally, the above preparations were added to 24-well plates for transfection experiments, 0.5 μg GFP mRNA / well, and continued to be cultured in an incubator at 37°C and 5% CO2 for 24 hours.

[0135] (3) Fluorescence and flow cytometry: 24 hours after transfection, the expression of GFP was observed and photographed under an inverted fluorescence microscope. The cells in the 24-well plate were then digested with trypsin and collected into a flow tube. The cells were centrifuged at 1200 rpm for 3 min, washed twice with 1 mL of pre-cooled phosphate buffered saline (PBS), and then resuspended in 200 μL of PBS.

[0136] Flow cytometry was used to detect the GFP-positive cell rate and mean fluorescence intensity (MFI).

[0137] The results are as follows Figure 4 As shown, the expression of GFP in DC2.4 cells was observed by inverted fluorescence microscopy. Figure 4 As shown in the microscopic observation diagram A, the expression of LPX and LPX+T groups was similar. According to the results of flow cytometry, the transfection efficiency was less than 20%. The expression of GFP in the T-LPX group prepared by co-loading tomatidine in liposomes was greatly improved, and the transfection efficiency reached about 60% ( Figure 4 B).

[0138] The above results indicate that directly mixing tomatidine with common mRNA preparations and then treating cells will not promote the expression of mRNA. The preparation composition of the present invention is used to co-deliver tomatidine and mRNA in a vector, and tomatidine can significantly enhance the expression of mRNA in cells.

[0139] 4. In vitro expression of T-LPX at different tomatidine concentrations

[0140] The specific operations are:

[0141] DC2.4 in the logarithmic growth phase were collected, resuspended and counted, and inoculated into 6-well plates at 8×104 / well, and cultured overnight in a cell incubator at 37°C and 5% CO2. The next day, LPX containing GFP-RNA and T-LPX containing different concentrations of tomatine (tomatine concentrations were 0.15mM, 0.45mM, 0.75mM, 1.2mM, and 1.5mM) were prepared according to the method in the above 1. Preparation of T-LPX, and 0.5μg GFP-mRNA / well was added to the six-well plate. After 24h of transfection, the expression of green fluorescent protein was observed under an inverted fluorescence microscope; then the cells in the 6-well plate were digested with trypsin and collected into a flow tube, centrifuged, and washed twice with pre-cooled PBS; 100μL PBS was added to resuspend the cells, and the percentage of GFP-positive cells and the mean fluorescence intensity (MFI) in live cells were detected by flow cytometry to analyze the cell transfection.

[0142] Results Figure 5 , the expression of GFP in DC2.4 cells was observed by inverted fluorescence microscopy. Figure 5 A is a microscope observation picture; according to the results of flow cytometry, such as Figure 5As shown in B, T-LPX containing 0.45 mM tomatidine has the best transfection effect on DC2.4. As the concentration of tomatidine in the liposome increases, the transfection efficiency and MFI first increase and then decrease. The above transfection results show that liposomes added with tomatidine can protect and promote the expression of GFP-mRNA on DC2.4, and the optimal concentration of tomatidine is 0.45 mM.

[0143] 5. Preparation stability study

[0144] The T-LPX with the optimal concentration of tomatidine of 0.45 mM in the above 4 was selected, and three batches were prepared for DC2.4 transfection experiments. The percentage and MFI of GFP-positive cells in living cells were detected by flow cytometry to investigate the batch stability of T-LPX;

[0145] The results are as follows Figure 6 As shown, three batches of prepared T-LPs were used to load GFP-mRNA for transfection experiments, and there was no significant difference in transfection efficiency and MFI, demonstrating the good reproducibility of the preparations during in vitro evaluation.

[0146] 6. Transfection stability study

[0147] The T-LPX prepared above and loaded with GFP-mRNA was placed in a 4°C refrigerator, and DC2.4 transfection experiments were performed on day 0, 3, 7, and 14, respectively. The percentage and MFI of GFP-positive cells in living cells were determined to investigate the transfection stability of T-LPX.

[0148] The results are as follows Figure 7 As shown, there was no significant difference in the transfection results on day 0, 3, and 7, and the transfection efficiency and MFI dropped sharply on day 14, indicating that T-LPX was stable at 4°C for 7 days.

[0149] 7. Antigen presentation of T-LPX BMDCs

[0150] BMDCs were collected on day 8, resuspended and counted, and seeded into 6-well plates at 5×105 / well, and cultured overnight in an incubator at 37°C and 5% CO2. The next day, LPX loaded with OVA-mRNA and T-LPX containing different concentrations of tomatidine (tomatidine concentrations were 0.15mM, 0.45mM, 0.75mM, 1.2mM, and 1.5mM) were prepared according to the method in 1. Preparation of T-LPX, and 1μg OVA-mRNA / well was added into 6-well plates. After incubation for 24 hours, cells in the 6-well plate were collected into flow cytometry tubes using a cell scraper, centrifuged, and washed twice with pre-cooled PBS; 100 μL PBS was added to resuspend the cells, 1 μL Anti-mouse CD16 / 32 was added, mixed and incubated at 4°C for 15 minutes, and then 1 μL PE-anti-mouse CD11c and APC-anti-mouse SIINFEKL / H-2Kb 25-D1.16 flow cytometry antibodies were added respectively, and incubated at 4°C in the dark for 40 minutes; after washing twice with PBS, the percentage of H-2Kb-positive cells in CD11c-positive cells was detected by flow cytometry to analyze the cellular antigen presentation.

[0151] Results Figure 8 ( Figure 8 A is the distribution map, Figure 8 B is the corresponding bar graph); T-LPX containing 0.45 mM tomatidine significantly enhanced antigen presentation ability compared with LPX (p<0.001), and the trend was consistent with DC2.4 transfection.

[0152] 9. Ability of T-LPX to activate DCs in vitro

[0153] Quantitative detection of CD11c by flow cytometry + CD80 in BMDCs + CD86 + The proportion of double-positive cells was used to investigate the expression of co-stimulatory molecules by T-LPX-induced DCs.

[0154] The experimental method is as follows:

[0155] (1) BMDCs cell plating: BMDCs were collected, resuspended and counted, and the cell suspension was adjusted to an appropriate concentration with 1640 complete medium. Then, 5×10 5 / 500 μL / well was inoculated into a 24-well plate, shaken evenly from side to side, and cultured overnight in a 37°C, 5% CO2 cell incubator.

[0156] (2) Preparation and administration: The next day, OVA mRNA-loaded preparations for BMDCs maturation and activation experiments were prepared according to the method in 1. Preparation of T-LPX, as well as T-LPX with different concentrations of tomatidine (tomatidine concentrations were 0.15 mM, 0.45 mM, 0.75 mM, 1.2 mM, and 1.5 mM). Subsequently, LPX and T-LPX were added to a 24-well plate at a rate of 1 μg OVA mRNA / well and cultured in an incubator at 37°C, 5% CO2 for 24 h.

[0157] (3) Staining and flow cytometry: After 24 hours of incubation, collect the cells in the 24-well plate into flow tubes, centrifuge at 1500 rpm for 5 minutes, wash the cells twice with pre-cooled 1 mL PBS, and remove the supernatant. Then add 100 μL PBS to each flow tube to resuspend the cells, add 1 μL Anti-mouse CD16 / 32, mix well, and incubate at 4°C for 15 minutes to block non-specific binding. Then add 1 μL PE-anti-mouse CD11c, APC-anti-mouse CD80 and FITC-anti-mouse CD86 flow antibodies to the corresponding flow tubes, and incubate at 4°C for 40 minutes. After incubation, wash the cells with 1 mL pre-cooled PBS, centrifuge at 1500 rpm for 5 minutes, and then add 300 μL PBS to mix well. Detect the mature activation state of BMDCs by flow cytometry.

[0158] The results are as follows Fig. 9 show( Fig. 9 A is the diagram of BMDCs maturation and activation. Fig. 9 (B is the corresponding bar graph). Compared with the Control group without mRNA drugs, the proportion of CD80+CD86+ double positive cells in LPX-treated BMDCs did not increase significantly, about 20% (p>0.05). However, the proportion of CD80+CD86+ double positive cells in T-LPX-treated BMDCs increased significantly by more than two times, close to 50% (p<0.001), especially in T-LPX with a tomatidine concentration of 0.45mM and above, the proportion of CD80+CD86+ double positive cells increased significantly.

[0159] This indicates that, compared with the incubation of LPX with BMDCs which cannot promote the maturation and activation of BMDCs, the preparation composition of the present invention co-loaded with tomatidine T-LPX can promote the maturation of BMDCs and is beneficial to the initiation of T cell immune response.

[0160] 10. Distribution and expression of T-LPX in vivo

[0161] Using Luc mRNA as a tool gene, T-LPX loaded with lipophilic carbocyanine dye (DID) and Luc mRNA was prepared to investigate the distribution and expression of T-LPX in various organs in vivo.

[0162] The lipid organic phase was added to prepare liposomes loaded with fluorescent dye, and then mixed with Luc mRNA to obtain DID-loaded LPX and T-LPX for investigating the uptake and expression of the preparation in vivo. C57BL / 6 mice were injected with the above preparation through the tail vein at a dose of 20μg Luc mRNA / mouse and 20μg DID / mouse, and PBS was injected at the same time as the negative control group Control. 6h after administration, 200μL of luciferase substrate solution (15mg / mL) was injected intraperitoneally. Euthanasia was performed 10min later, and the liver, spleen, and lungs where the preparation was mainly distributed were quickly dissected, and the DID fluorescence intensity and bioluminescence intensity of each organ were detected in the mouse imager.

[0163] like Fig.10 Mouse imaging and Fig.11 The results of quantitative detection show that Fig.10 It can be seen that the preparation composition of the present invention is widely distributed in the liver, spleen and lungs, and is mainly expressed in the lungs; Fig.11 It can be seen that compared with LPX, T-LPX has a stronger distribution in various organs, especially in the lungs; and the expression of T-LPX in the lungs is also stronger than that of LPX.

[0164] The above results verified that tomatidine can enhance the distribution and expression of T-LPX in vivo.

[0165] 11. Effect of tomatidine on the anti-tumor effect of T-LPX vaccine

[0166] Construction of E.G7-OVA tumor model:

[0167] OVA-mRNA was selected as a model antigen to evaluate the effect of tomatidine on the activation of specific T cell immune response and anti-tumor activity of T-LPX tumor vaccine. The effect of tomatidine on enhancing the antigen signal and co-stimulatory signal of T-LPX tumor vaccine in vivo was also investigated.

[0168] (1) Establishment of E.G7-OVA tumor model

[0169] Collect E.G7-OVA cells in the logarithmic growth phase, wash the cells twice with sterile pre-cooled PBS, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells with sterile PBS, and adjust the cell density to 1×10 750 μL of E.G7-OVA cells were subcutaneously inoculated in the right rib of 7-8 week old male C57BL / 6 mice, and the growth status of the mice and the size of the subcutaneous tumor were observed. Small tumors visible to the naked eye were formed 7 days after inoculation and could be used for subsequent experiments.

[0170] (2) Tumor immunotherapy regimen and process monitoring

[0171] Experimental groups: The day of tumor inoculation was designated as Day 0. Small tumors visible to the naked eye were formed 7 days after E.G7-OVA cell inoculation. On Day 7, the tumor-bearing mice were randomly divided into groups and numbered with ear tags.

[0172] Preparation and administration: LPX / OVA and T-LPX / OVA loaded with OVA mRNA were prepared, and a negative control preparation T-LPX@Luc with no therapeutic mRNA was prepared. Tumor-bearing mice were injected with 180 μL of vaccine preparation, i.e. 30 μg OVA mRNA per mouse, through the tail vein each time, and PBS was injected into the tail vein as a negative control group Control. Immunization was performed once on Day 7, Day 10, and Day 15, for a total of 3 immunotherapy sessions.

[0173] Tumor volume monitoring: Starting from Day 7, the tumor volume was measured every three days and the tumor growth curve was drawn. Tumor volume calculation formula: V (mm 3 )=a×b 2 / 2, where a is the maximum diameter of the tumor (mm) and b is the length of the radial line perpendicular to a (mm).

[0174] The results are as follows Fig.12 As shown, the tumor volume of mice in the Control group injected with PBS and the mRNA preparation T-LPX / Luc group with no therapeutic effect increased day by day. The mRNA vaccine preparations LPX / OVA and T-LPX / OVA loaded with specific antigen OVA mRNA can significantly inhibit tumor growth, among which T-LPX / OVA has a better tumor inhibition effect and the tumor tends to shrink. On the 25th day, the mice were killed, the tumors were taken and weighed, and the average tumor weight of the T-LPX / OVA group was significantly lighter than that of the LPX / OVA group. The above results show that co-loading tomatine can further enhance the anti-tumor activity of mRNA vaccines and inhibit tumor growth.

[0175] (3) T cell immune response detection

[0176] Preparation of single cell suspension: On Day 25, mice were killed and spleens were taken to prepare single cell suspensions. Specifically, appropriate amount of spleen tissue was placed in a 70μm filter, and then the filter was placed in a 6-well plate, 3-5mL PBS was added to cover the tissue, and then the spleen tissue was ground with a 5mL syringe head, and the single tissue cell suspension was collected after rinsing the filter into a flow tube. Centrifuge at 1200rpm for 5min, discard the supernatant, add 2mL of pre-cooled red blood cell lysis buffer, blow the cells evenly, and lyse on ice for about 30min to remove red blood cells. Centrifuge at 1200rpm for 5min, discard the supernatant, wash the cells twice with 2mL PBS, and finally resuspend the cells with 100μL PBS for flow antibody staining.

[0177] Antibody staining: First, add 1 μL anti-mouse CD16 / 32 to 100 μL single cell suspension and incubate at 4°C for 15 min to block nonspecific binding. Then add 1 μL APC anti-mouse CD8a and PE anti-mouse SIINFEKL / H-2Kb OVA tetramer flow cytometry antibodies, vortex to mix, and incubate at 4°C in the dark for about 40 min. Finally, add 1 mL PBS to wash the cells twice, centrifuge at 1200 rpm for 5 min, add 200 μL PBS to resuspend the cells, and detect OVA-specific CTLs in the spleen by flow cytometry.

[0178] Fig.13 The results of specific T cell assay showed that ( Fig.13 A is a cell diagram, Fig.13 B is the corresponding bar graph). Both LPX / OVA and T-LPX / OVA can significantly induce the production of OVA-specific CTLs in the spleen. The proportion of OVA-specific CTLs in mice immunized with T-LPX / OVA is about 7%, which is significantly better than that in mice immunized with LPX / OVA. This indicates that the T-LPX / OVA vaccine preparation co-loaded with tomatidine can induce a stronger T cell immune response, produce more specific CTLs, and enhance the anti-tumor activity of the tumor vaccine.

[0179] (4) Antigen presentation by DCs in vivo

[0180] Based on the above experiments, we further investigated the antigen presentation and maturation activation of DCs in vivo after T-LPX / OVA immunization. First, C57BL / 6 mice were injected with LPX / OVA and T-LPX / OVA loaded with 30 μg OVA mRNA via the tail vein. 24 hours later, single cell suspensions were prepared from spleen, lymph nodes, and lungs for flow cytometry antibody staining.

[0181] Antigen presentation of DCs in vivo: 1 μL anti-mouse CD16 / 32 was added to the single cell suspension of spleen and lymph nodes, and incubated at 4°C for 15 minutes to block nonspecific binding. Then 1 μL PE anti-mouse CD11c and APC-anti-mouse SIINFEKL / H-2Kb flow cytometry antibodies were added, vortexed and incubated at 4°C in the dark for about 40 minutes. Finally, 1 mL PBS was added to wash the cells twice, centrifuged at 1200 rpm for 5 minutes, and 200 μL PBS was added to resuspend the cells. The percentage of H-2Kb positive cells in CD11c positive cells was detected by flow cytometry to analyze the antigen presentation of DCs in spleen and lymph nodes.

[0182] (5) Maturation and activation of DCs in vivo

[0183] Add 1 μL anti-mouse CD16 / 32 to the single cell suspension of spleen and lung, and incubate at 4°C for 15 minutes to block nonspecific binding. Then add 1 μL PE anti-mouse CD11c, FITC anti-mouse CD80 and APC anti-mouse CD86 flow cytometry antibodies, vortex and mix, and incubate at 4°C in the dark for about 40 minutes. Finally, add 1 mL PBS to wash the cells twice, centrifuge at 1200 rpm for 5 minutes, add 200 μL PBS to resuspend the cells, and detect the percentage of CD80 and CD86 double-positive cells in CD11c-positive cells by flow cytometry to analyze the maturation and activation of DCs in spleen and lung.

[0184] 24 h after intravenous injection of LPX / OVA and T-LPX / OVA, single cell suspensions were extracted from spleen and lymph nodes of mice for antibody staining. The flow cytometry results are shown in Fig.14 As shown in (A is the spleen result, B is the lung result), T-LPX / OVA can produce more specific OVA antigen peptide signals in both spleen and lymph nodes. Fig.15 (A is the spleen result, B is the lymph node result) As shown, compared with LPX / OVA, T-LPX / OVA can induce more DCs maturation and activation in both the spleen and lungs. This shows that DCs in T-LPX / OVA-immunized mice provide more co-stimulatory cells (CD80 and CD86) to assist antigen signals, initiate stronger specific T cell responses, and enhance their anti-tumor activity.

[0185] Example 2 Tomatine enhances the activity of mRNA COVID-19 vaccine based on lipid nanoparticle delivery vector LNP

[0186] 1. Preparation of T-NPX

[0187] like Fig.16 As shown, lipid nanoparticles (T-NPX) co-loaded with tomatidine and mRNA were prepared using microfluidic technology.

[0188] (1) Solution preparation: mRNA was prepared with 10 mM pH 6.0 citric acid-sodium citrate buffer (prepared with RNase-free water) to an appropriate concentration as the aqueous phase for later use; then, the organic phase was prepared according to the mass ratio of cationic lipid to mRNA of 15:1, the mass ratio of tomatidine to mRNA of 3:1, and the molar ratio of MIC5, DOPE, Chol and DMG-PEG2000 of 35:16:46.5:2.5. Specifically, the above-mentioned liposomes and tomatidine were dissolved in ethanol with a volume of 1 / 3 of the aqueous phase as the organic phase.

[0189] (2) Preparation by microfluidics: The organic phase and aqueous phase are passed through a microfluidic device and mixed evenly to form lipid nanoparticles, namely T-NPX; NPX is prepared by the same method without adding tomatidine to the organic phase. The microfluidic process parameters are as follows: the volume ratio of the organic phase to the aqueous phase is 1:3, and the flow rate is 12 mL / min.

[0190] (3) Ultrafiltration: 10 mM citric acid-sodium citrate buffer at pH 6.0 containing 10% sucrose was prepared with RNase-free water. The lipid nanoparticle preparation was diluted 25 times with this buffer (the ethanol concentration in the preparation was 1%), and then the preparation was concentrated to the initial volume by ultrafiltration cup to reduce the concentration of ethanol in the preparation. The ultrafiltration process parameters were as follows: air pressure 0.2 MPa, filter membrane 100 kDa, rotation speed 100-200 rpm.

[0191] 2. Pharmaceutical investigation of T-NPX

[0192] Take an appropriate amount of NPX and T-NPX formulation solutions and dilute them 10 times with purified water, and use Malvern laser particle size analyzer to detect the formulation properties such as particle size, particle size distribution and zeta potential. Each sample was measured three times in parallel, and the measurement temperature was 25°C.

[0193] Depend on Fig.17 (A is the particle size and PDI diagram, B is the potential diagram) It can be seen that the addition of tomatidine does not affect the particle size and potential of the final lipid nanoparticles. The particle size of NPX and T-NPX is about 110nm, the PDI is less than 0.3, and the potential is neutral.

[0194] 3. In vitro expression of T-NPX

[0195] The transfection efficiency of NPX and T-NPX in DC2.4 cells was detected.

[0196] The transfection method is as follows:

[0197] (1) Cell plating: DC2.4 cells in the logarithmic growth phase were collected by trypsin digestion, resuspended in DMEM complete medium, and counted. Then, 1×10 5 500 μL / well were inoculated into a 24-well plate, shaken evenly, and cultured overnight in a 37°C, 5% CO2 cell incubator.

[0198] (2) Preparation and administration: Lipid nanoparticles (T-NPX) co-loaded with GFP mRNA and tomatidine were prepared using microfluidic technology; ordinary lipid nanoparticles (NPX) were prepared in the same way. At the same time, the tomatidine compound solution was physically mixed with the prepared NPX to set up the NPX+T group. Finally, the above preparations were added to 24-well plates for transfection experiments, 0.5 μg GFP mRNA / well, and continued to be cultured in an incubator at 37°C and 5% CO2 for 24 hours.

[0199] (3) Fluorescence and flow cytometry: 24 hours after transfection, the expression of GFP was observed and photographed under an inverted fluorescence microscope. The cells in the 24-well plate were then digested with trypsin and collected into a flow tube. The cells were centrifuged at 1200 rpm for 3 min, washed twice with 1 mL of pre-cooled PBS, and then resuspended in 200 μL of PBS. The GFP-positive cell rate and mean fluorescence intensity (MFI) were detected by flow cytometry.

[0200] The results are as follows Fig.18 As shown in (A is a microscope observation diagram; B is a cytometry detection data diagram), the transfection results show that the MFI of T-NPX is higher than that of NPX. At the same time, the transfection ability of NPX+T and NPX is comparable.

[0201] 4. Investigation of the ability of T-NPX to activate DCs in vitro

[0202] Co-loading tomatidine and antigen mRNA into lipid nanoparticles activates the ability of DCs to provide co-stimulatory signals.

[0203] (1) BMDCs cell plating: BMDCs were collected, resuspended and counted, and the cell suspension was adjusted to an appropriate concentration with 1640 complete medium. Then, 5×10 5 / 500 μL / well was inoculated into a 24-well plate, shaken evenly from side to side, and cultured overnight in a 37°C, 5% CO2 cell incubator.

[0204] (2) Preparation and administration: NPX and T-NPX loaded with OVA mRNA were prepared using microfluidics technology, and then 0.5 μg of OVA mRNA / well was added to a 24-well plate and cultured in an incubator at 37° C. and 5% CO 2 for 24 h.

[0205] (3) Staining and flow cytometry: After 24 hours of incubation, collect the cells in the 24-well plate into flow tubes, centrifuge at 1500 rpm for 5 minutes, wash the cells twice with pre-cooled 1 mL PBS, and remove the supernatant. Then add 100 μL PBS to each flow tube to resuspend the cells, add 1 μL Anti-mouse CD16 / 32, mix well, and incubate at 4°C for 15 minutes to block non-specific binding. Then add 1 μL PE-anti-mouse CD11c, APC-anti-mouse CD80 and FITC-anti-mouse CD86 flow antibodies to the corresponding flow tubes, and incubate at 4°C for 40 minutes. After incubation, wash the cells with 1 mL pre-cooled PBS, centrifuge at 1500 rpm for 5 minutes, and then add 300 μL PBS to mix well. Detect the mature activation state of BMDCs by flow cytometry.

[0206] The results are as follows Fig.19 (A is the cell image, B is the corresponding bar graph) shows that compared with the control and NPX, T-NPX co-delivered by tomatidine also further promoted the ability of BMDCs to provide more co-stimulatory signals for maturation and activation.

[0207] 5. In vivo expression of T-NPX

[0208] NPX and T-NPX loaded with Luc mRNA were injected intramuscularly at a dose of 20 μg mRNA per mouse. 6h and 24h later, 200 μL (15 mg / mL) of d-luciferin potassium solution were injected intraperitoneally, respectively. The bioluminescence intensity of the mice was detected using a small animal imaging device.

[0209] like Fig. 20 As shown in (A is the in vivo imaging diagram, B is the data result diagram), tomatidine significantly enhanced the expression of T-NPX loaded with Luc mRNA in mice.

[0210] 6. Distribution and expression of T-LPX in vivo

[0211] The ability of tomatidine to enhance mRNA vaccines to prevent infectious diseases was studied using NPX and T-NPX loaded with mRNA encoding the SARS-CoV-2 Delta variant spike protein. BALB / c mice (n=6) in each group were injected intramuscularly with a high dose (30μg mRNA) and a low dose (10μg mRNA) at week 0 and week 2, respectively. ELISA was used to detect the RBD-specific IgG response of SARS-CoV-2 in sera collected at weeks 2, 4, 6, 8, and 10. At the same time, the ability of NPX and T-NPX to induce cellular immune responses in mice was detected by flow cytometry and enzyme-linked immunospot (ELISpot).

[0212] (1) Antibody detection: Enzyme-linked immunosorbent assay (ELISA) was used to detect specific antibody titers. The ELISA plate was coated with SARS-COV-2delta, wild-type, and omicron RBD at a final concentration of 1 μg / mL, incubated overnight at 4°C, washed four times with washing buffer, and then blocked with 2% bovine serum albumin (BSA) at 25°C for 4 hours. After washing twice with washing buffer, serially diluted serum samples were added and incubated overnight at 4°C. After washing four times with washing buffer, HPR-conjugated goat anti-mouse IgG (1:50000 dilution) was added and incubated at 25°C for 2 hours. After washing four times with washing buffer, trimethylborane (TMB) solution was added and incubated for 30 minutes, and finally the reaction was stopped with 2M sulfuric acid. The absorbance was measured at 450 nm using an enzyme reader (PerkinElmer, USA).

[0213] (2) T cell immune response detection: Prepare single cell suspension of spleen and lymph nodes, add 1 μL anti-mouse CD16 / 32 to 100 μL single cell suspension, incubate at 4°C for 15 min to block nonspecific binding. Then add 1 μL APC anti-mouse CD8a and FITC anti-mouse CD4 flow cytometry antibodies respectively, vortex to mix, and incubate at 4°C in the dark for about 40 min. Finally, add 1 mL PBS to wash the cells twice, centrifuge at 1200 rpm for 5 min, add 200 μL PBS to resuspend the cells, and detect CD4 by flow cytometry. + and CD8 + proportion.

[0214] (3) ELISpot assay: ELISpot detection of IFN-γ of splenocytes was performed using the mouse IFN-γ ELISpotPLUS kit instructions. The 96-well plate pre-coated with IFN-γ antibody was washed with PBS and incubated with RPMI 1640 culture medium coated with 10% PBS at room temperature for 30 minutes. Splenocytes were then stimulated with a pool of SARS-COV-2delta variant peptides (2 μg / ml single peptide) and added to the plate (5×10 per well). 5 Cells). Culture at 37°C for 48 h, remove the cells, and wash with PBS 5 times. Subsequently, incubate with biotinylated anti-IFN-γ antibody at room temperature for 2 h. Expose the spots after adding substrate solution and rinse with tap water. Dry naturally in a cool place, and read the number of spots with an ELISpot reader.

[0215] like Fig.21As shown, at low doses, there was no significant difference in IgG titers between NPX and T-NPX. However, at high doses, the IgG titers of the T-NPX group against SARS-CoV-2 and its variants (including delta type, wild-type type, and omicron type) were significantly higher than those of the NPX group. The above results suggest that tomatidine can promote stronger protective antibody responses induced by SARS-CoV-2 mRNA vaccines.

[0216] Fig. 22 (A is the imaging image, B is the data bar graph) The T cell detection results show that T-NPX induces an increase in CD4+T cells and CD8+T cells. Fig.23 (A is the observed image, B is the data bar graph) As shown, due to the effect of tomatidine, the T cells secreting IFN-γ in T-NPX-immunized mice were significantly more than those in NPX-immunized mice. These results indicate that the addition of tomatidine enhances the activity of SARS-CoV-2 mRNA vaccines and can induce strong humoral and Th1 T cell responses.

[0217] It is worth noting that the specific features, structures, materials or characteristics described in this specification can be combined in any one or more examples in an appropriate manner. In addition, those skilled in the art can combine the features of different examples and different embodiments described in this specification without contradiction.

Claims

1. Use of glycoside compounds in nucleic acid drugs to promote nucleic acid expression or DCs maturation and activation, characterized in that: The glycoside compound is one or more selected from tomatine and its derivatives, tomatine analogs and their derivatives, solanine and its derivatives, solanine and its derivatives, chaconine and its derivatives, and solanine and its derivatives.

2. A preparation composition, characterized in that: It comprises a glycoside compound, a nucleic acid drug and a delivery vector; wherein the glycoside compound is selected from one or more of tomatine and its derivatives, tomatine analogs and their derivatives, solanine and its derivatives, solanine and its derivatives, chaconine and its derivatives, and solanine and its derivatives.

3. The use according to claim 1 or the formulation composition according to claim 2, characterized in that: The glycoside compound is tomatidine and its derivatives.

4. The preparation composition according to claim 2, characterized in that in, The nucleic acid drug is selected from one or more of DNA, ASO, siRNA, miRNA, mRNA, ribozyme, and nucleic acid aptamer.

5. The preparation composition according to claim 4, characterized in that: The nucleic acid drug is mRNA; the mRNA encodes one or more of a viral antigen protein, a protein supplementation therapeutic protein, a tumor antigen protein, a gene editing tool protein or a cytokine.

6. The preparation composition according to claim 2, characterized in that The delivery carrier co-carries a glycoside compound and a nucleic acid drug; wherein the mass ratio of the glycoside compound to the nucleic acid drug is 0.02:1 to 10:

1.

7. The preparation composition according to claim 2, characterized in that The delivery carrier is selected from one or more of lipid nanoparticles, PLGA nanoparticles, micelles, liposomes, core-shell nanoparticles or polymer nanoparticles.

8. The preparation composition according to claim 7, characterized in that The delivery carrier lipid nanoparticles comprise one or more of cationic lipids, neutral phospholipids, steroids and polyethylene glycol lipids; wherein the cationic lipid is selected from the following structures of MIC1, MIC2, MIC3, MIC4, MIC5 or MIC6: The neutral phospholipid is selected from one or more of DOPE, DSPC, DOPC, DSPE, DMPC, DMPE, DPPC, DPPE, DEPC, HSPC or POPC; the molar ratio of the cationic lipid to the neutral phospholipid is preferably 0.1:1 to 20:1; preferably 1:1 to 2:1; The steroid is selected from cholesterol, sitosterol, soya sterol, lanosterol, ergosterol or a combination thereof; preferably, the molar ratio of the cationic lipid to the steroid is 1:10 to 10:1; preferably 1:2 to 2:1; The polyethylene glycol lipid is selected from DMG-PEG, DSPE-PEG or a combination thereof; preferably, the molar ratio of the cationic lipid to the polyethylene glycol lipid is 5:1 to 100:1; preferably 10:1 to 20:

1.

9. The formulation composition according to claim 2, characterized in that: The method comprises tomatidine, mRNA, MIC5, DOPE, cholesterol or DMG-PEG2000; wherein the mass ratio of tomatidine to mRNA is 0.5:1-10:1, and the mass ratio of MIC5 to mRNA is 15:

1.

10. The preparation composition according to claim 7, characterized in that The liposomes contain cationic lipids and steroids; wherein the cationic lipids are selected from one or more of DODAC, DDAB, DOTAP, DOTMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DOAP, DOPE, DODAP, and DMRIE; and the steroids are selected from one or more of cholesterol, sitosterol, soysterol, lanosterol, and ergosterol.

11. Use of the preparation composition according to any one of claims 2, 4 to 10 in the preparation of mRNA drugs for promoting mRNA expression or promoting DCs maturation and activation.

12. The use according to claim 1 or 11, characterized in that The formulation composition administers a dosage of about 0.001 mg / kg to about 10 mg / kg of the nucleic acid drug to the mammal; preferably, the mammal is a human; further, the dosage of the glycoside compound is 0.2 to 10 times that of the nucleic acid drug.

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