Hepatitis synergistic therapeutic ionizable lipid nanoparticles as well as preparation method and application thereof

Abstract

The invention provides a co-therapeutic ionizable lipid nanoparticle for hepatitis as well as a preparation method and application of the co-therapeutic ionizable lipid nanoparticle for hepatitis, and the co-therapeutic ionizable lipid nanoparticle for hepatitis comprises ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid, a phospholipid polyethylene glycol derivative and a siRNA (small interfering ribonucleic acid) medicine, the co-therapeutic ionizable lipid nanoparticle for hepatitis has dual effects, can simultaneously realize targeted delivery and treatment effects, improves the bioavailability and biocompatibility of the drug, and has a synergistic curative effect with the siRNA drug, so that the treatment effect on liver inflammation diseases is remarkably improved.

Description

[Technical field]

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a hepatitis synergistic therapeutic ionizable lipid nanoparticle and a preparation method and application thereof. [Background technology]

[0002] Hepatitis refers to inflammation of the liver caused by various factors (such as drugs, alcohol, viruses, poisoning, etc.), which lead to abnormalities in the structure and function of liver cells in the body, and increased concentrations of tumor necrosis factor-α (TNF-α) inflammatory factors, whose overproduction is closely related to the pathogenesis of hepatitis. In the current clinical setting, anti-TNF-α immunotherapy using TNF-α monoclonal antibodies, receptors or inhibitors provides a basis for the treatment of liver inflammation. However, these anti-TNF-α therapies are often costly and autoimmune to antibodies. Therefore, a new method is needed to mediate the release of TNF-α and treat liver inflammation. Small interfering RNA (siRNA) drugs can downregulate cytokines in activated macrophages (RAW 264.7) through RNA interference (RNAi), thereby inhibiting hepatitis. However, for different diseases, siRNA drugs need to specifically reach the target organ to play a role and inhibit gene expression, but they will be degraded by nucleases in the body if they enter the body directly. At the same time, since siRNA drugs themselves are negatively charged, they will repel cell membranes, resulting in poor therapeutic effects of siRNA drugs. In view of the limitations of siRNA drug applications, it is necessary to establish a carrier with protective and delivery properties to maximize the therapeutic effect of siRNA drugs.

[0003] At present, the common vectors in clinical applications are mainly viral vectors and non-viral vectors. Viral vectors are often limited by the inability to repeat administration, low specificity and high cost. Non-viral vectors can support repeated administration and instantaneous administration, and are low in cost. Among them, lipid nanoparticles can efficiently load siRNA drugs and effectively deliver siRNA drugs to the cytoplasm, achieve endosomal escape, and improve the therapeutic effect of siRNA drugs.

[0004] The use of lipid nanoparticles to deliver siRNA drugs into the body can effectively treat liver inflammation by exerting the gene silencing effect of siRNA drugs. However, existing lipid nanoparticles can only protect and deliver siRNA drugs and have no effect on the disease itself. The art needs to provide an ionizable lipid nanoparticle with synergistic therapeutic properties, so that the lipid nanoparticles can simultaneously achieve targeted delivery and therapeutic effects, improve the bioavailability and biocompatibility of the drug, and synergize with the siRNA drug to exert therapeutic effects, and is of great significance for developing new functions of lipid nanoparticles.

[0005] Therefore, it is necessary to study a hepatitis synergistic therapeutic ionizable lipid nanoparticle and its preparation method and application to address the shortcomings of the existing technology and to solve or alleviate one or more of the above problems. [Summary of the invention]

[0006] In view of this, the present invention provides a hepatitis synergistic therapeutic ionizable lipid nanoparticle and a preparation method and application thereof. The hepatitis synergistic therapeutic ionizable lipid nanoparticle has dual functions, can simultaneously achieve targeted delivery and therapeutic effects, improve the bioavailability and biocompatibility of drugs, synergistically exert therapeutic effects with siRNA drugs, and significantly improve the therapeutic effect on liver inflammatory diseases.

[0007] On the one hand, the present invention provides a hepatitis synergistic therapeutic ionizable lipid nanoparticle, wherein the hepatitis synergistic therapeutic ionizable lipid nanoparticle comprises ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid, phospholipid polyethylene glycol derivative and siRNA drug.

[0008] According to the aspects and any possible implementations described above, an implementation is further provided, wherein the ionizable lipids include one or more of 4-(N,N-dimethylamino)butyric acid (dialene) methyl ester, 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecanyloxy)hexyl]amino]-octanoate and ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0009] According to the aspects and any possible implementation described above, an implementation is further provided, wherein the auxiliary lipid includes one or more of 1,2-distearoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 2-oleoyl-1-palmitoyltinglycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dierucoyl-sn-glycero-3-phosphocholine.

[0010] According to the above aspects and any possible implementation, an implementation is further provided, wherein the hepatitis therapeutic lipid is 1,2-dilauroyl-sn-glycero-3-phosphocholine.

[0011] According to the above aspects and any possible implementation, an implementation is further provided, wherein the phospholipid polyethylene glycol derivative is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000.

[0012] According to the above aspects and any possible implementation, a method for preparing hepatitis synergistic therapeutic ionizable lipid nanoparticles is further provided, which is used to prepare the hepatitis synergistic therapeutic ionizable lipid nanoparticles, and the preparation method comprises the following steps:

[0013] S1: Weigh a certain mass of ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid and phospholipid polyethylene glycol derivative respectively, dissolve them in ethanol respectively, mix them evenly in a certain molar ratio, and obtain an organic phase;

[0014] S2: dissolving the siRNA drug in DEPC water at a certain concentration, and mixing it with the acidic buffer solution at a certain volume ratio to obtain an aqueous phase;

[0015] S3: adding a certain volume of the organic phase to the aqueous phase according to a certain mass ratio of the lipid mixture and the siRNA drug in the organic phase, mixing, and incubating to obtain a mixed solution;

[0016] S4: The mixed solution is added into a 3500Da dialysis bag and dialyzed in 1× phosphate buffer for a certain period of time to obtain hepatitis synergistic therapeutic ionizable lipid nanoparticles.

[0017] According to the aspects and any possible implementation described above, an implementation is further provided, wherein the molar ratio of the ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid and phospholipid polyethylene glycol derivative in S1 is 50: (0-15): 38.5: (0.1-15): 1.5.

[0018] According to the aspects and any possible implementations described above, an implementation is further provided, wherein the mass concentration of the siRNA aqueous solution in S2 is 0.1-3.0 μg / μL, the pH of the acidic buffer solution is less than 7.4, and the volume ratio of the siRNA aqueous solution to the acidic buffer solution is 1:(1-30).

[0019] According to the above aspects and any possible implementation, an implementation is further provided, wherein the mass ratio of the five lipid mixtures to the siRNA drug in S3 is (1-60):1.

[0020] According to the aspects and any possible implementations described above, a further application of hepatitis synergistic therapeutic ionizable lipid nanoparticles is provided, wherein the application specifically comprises: synergistically treating hepatitis with the hepatitis synergistic therapeutic ionizable lipid nanoparticles and siRNA drugs.

[0021] Compared with the prior art, the present invention can achieve the following technical effects:

[0022] (1) The hepatitis synergistic therapeutic ionizable lipid nanoparticles of the present invention solve the problem of siRNA drug delivery. The ionizable lipids are used to encapsulate the siRNA drug through the electrostatic interaction between the ionizable lipids and the siRNA drug, and the siRNA drug is delivered to cells together with auxiliary lipids, cholesterol, hepatitis therapeutic lipids and phospholipid polyethylene glycol derivatives;

[0023] (2) The hepatitis synergistic therapeutic ionizable lipid nanoparticles of the present invention efficiently deliver siRNA drugs to macrophages and downregulate the expression of pathogenic proteins;

[0024] (3) The hepatitis synergistic therapeutic ionizable lipid nanoparticles of the present invention have dual therapeutic effects. On the one hand, the delivered siRNA drugs can inhibit the overexpression of cytotoxic factors that cause liver inflammation; on the other hand, the hepatitis therapeutic lipids can also regulate the production of TNF-α, reduce liver toxicity, and effectively treat liver inflammation. It also provides new ideas for the functional research of lipid nanoparticles, which has important research significance.

[0025] Of course, any product implementing the present invention does not necessarily need to achieve all of the above-mentioned technical effects at the same time.

Brief Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is a particle size distribution diagram of ionizable lipid nanoparticles for synergistic treatment of hepatitis provided by one embodiment of the present invention;

[0028] Figure 2 This is a result diagram of the embedding rate of ionizable lipid nanoparticles for synergistic treatment of hepatitis provided by one embodiment of the present invention;

[0029] Figure 3 One embodiment of the present invention provides a graph showing the cytotoxicity results of hepatitis synergistic therapeutic ionizable lipid nanoparticles;

[0030] Figure 4 One embodiment of the present invention provides a graph of the in vivo biodistribution results of hepatitis synergistic therapeutic ionizable lipid nanoparticles at the animal level;

[0031] Figure 5 This is a diagram showing the therapeutic effect of hepatitis synergistic therapeutic ionizable lipid nanoparticles on animals after treatment according to an embodiment of the present invention;

[0032] Figure 6An embodiment of the present invention provides a graph showing the effect of hepatitis synergistic therapeutic ionizable lipid nanoparticles on liver function in an animal hepatitis model. [Specific implementation method]

[0033] In order to better understand the technical solution of the present invention, the embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0034] It should be clear that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "said" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0036] The invention provides a hepatitis synergistic therapeutic ionizable lipid nanoparticle, which comprises ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid, phospholipid polyethylene glycol derivative and siRNA drug.

[0037] In the present invention, the head of the ionizable lipid molecule carries a positive charge and can encapsulate the negatively charged siRNA drug. By changing its charge state, the siRNA drug is safely delivered into the cell, achieving endosomal escape, and the siRNA drug is released into the cytoplasm to exert a gene silencing effect.

[0038] Preferably, the ionizable lipid is selected from one of 4-(N,N-dimethylamino)butyric acid (diaoleyl) methyl ester (Dlin-MC3-DMA), 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecanyloxy)hexyl]amino]-octanoate (SM-102), and ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0039] In the present invention, the auxiliary lipids can spontaneously organize into a lipid bilayer, and the higher phase transition temperature can enhance the membrane stability of the ionizable lipid nanoparticles, destroy the structure of the endosome, and improve the delivery efficiency of the siRNA drug.

[0040] Preferably, the auxiliary lipid is selected from one of 1,2-distearoyl-sn-glycero-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-oleoyl-1-palmitoyltin glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC).

[0041] In the present invention, the hepatitis therapeutic lipid can inhibit the release of the cytokine TNF-α, alleviate the liver toxicity caused by excessive production of TNF-α, give the ionizable lipid nanoparticles therapeutic function, work together with the siRNA drug, and improve the drug utilization.

[0042] Preferably, the hepatitis therapeutic lipid is selected from 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC).

[0043] In the present invention, the phospholipid polyethylene glycol derivative provides an external polymerization layer for the ionizable lipid nanoparticles to prevent serum protein adsorption and uptake by the mononuclear phagocyte system, thereby prolonging the circulation time in the body. On the other hand, it can also prevent the aggregation of the ionizable lipid nanoparticles during storage and in the blood.

[0044] Preferably, the phospholipid polyethylene glycol derivative is selected from 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000).

[0045] The present invention also provides a method for preparing hepatitis synergistic therapeutic ionizable lipid nanoparticles, which is used to prepare the hepatitis synergistic therapeutic ionizable lipid nanoparticles, and the preparation method comprises the following steps:

[0046] S1: Weigh a certain mass of ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid and phospholipid polyethylene glycol derivative respectively, dissolve them in ethanol respectively, mix them evenly in a certain molar ratio, and obtain an organic phase;

[0047] S2: dissolving the siRNA drug in DEPC water at a certain concentration, and mixing it with the acidic buffer solution at a certain volume ratio to obtain an aqueous phase;

[0048] S3: adding a certain volume of the organic phase to the aqueous phase according to a certain mass ratio of the lipid mixture and the siRNA drug in the organic phase, mixing, and incubating to obtain a mixed solution;

[0049] S4: The mixed solution is added to a 3500Da dialysis bag and dialyzed in 1× phosphate buffer (PBS, pH=7.4) for a certain period of time to obtain hepatitis synergistic therapeutic ionizable lipid nanoparticles.

[0050] Preferably, the mass concentration of the five lipid molecules described in S1 in the ethanol solution is 0.1-75 μg / μL, including but not limited to 0.1 μg / μL, 0.5 μg / μL, 1 μg / μL, 2 μg / μL, 3 μg / μL, 4 μg / μL, 5 μg / μL, 10 μg / μL, 25 μg / μL, 40 μg / μL, 55 μg / μL, 65 μg / μL or 75 μg / μL, preferably 1-65 μg / μL, and more preferably 1-55 μg / μL.

[0051] Preferably, the molar ratio of the ionizable lipid: auxiliary lipid: cholesterol: hepatitis therapeutic lipid: phospholipid polyethylene glycol derivative in S1 is 50: (0-15): 38.5: (0.1-15): 1.5, including but not limited to 50: 0.1: 38.5: 0.1: 1.5, 50: 0.5: 38.5: 0.5: 1.5, 50: 1: 38.5: 1: 1.5, 50: 5: 38.5: 5: 1.5, 50: 10: 38.5: 10: 1.5 or 50: 15: 38.5: 15: 1.5, preferably 50: (0-10): 38.5: (0.5-10): 1.5, and further preferably 50: (0-10): 38.5: (1-10): 1.5.

[0052] Preferably, the mass concentration of the siRNA aqueous solution in S2 is 0.1-3.0 μg / μL, including but not limited to 0.1 μg / μL, 0.2 μg / μL, 0.4 μg / μL, 0.6 μg / μL, 0.8 μg / μL, 1.0 μg / μL, 1.5 μg / μL, 2.0 μg / μL, 2.5 μg / μL or 3.0 μg / μL, preferably 0.2-1.5 μg / μL, and further preferably 0.4-0.8 μg / μL.

[0053] Preferably, the pH of the acidic buffer solution in S2 is less than 7.4, including but not limited to 7.0, 6.6, 6.4, 6.0, 5.6, 5.4, 5.0, 4.6, 4.4, 4.0, 3.6, 3.4, 3.0, 2.6, 2.4 or 2.0, preferably 2 to 7.0, further preferably 2 to 6.0, and more preferably 3 to 5.6.

[0054] Preferably, the acidic buffer solution in S2 includes any one of glycine-hydrochloric acid buffer solution, phthalic acid-hydrochloric acid buffer solution, disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate-sodium hydroxide buffer solution or Tris-hydrochloric acid buffer solution, preferably any one of disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, citric acid-sodium citrate buffer solution or disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution.

[0055] Preferably, the volume ratio of the siRNA aqueous solution and the acidic buffer in S2 is 1:(1-30), including but not limited to 1:2, 1:3.5, 1:5, 1:8, 1:10, 1:15, 1:20, 1:25 or 1:30, preferably 1:(1-15), and further preferably 1:(2-5).

[0056] Preferably, the mass ratio of the five lipid mixtures to the siRNA drug in S3 is (1-60):1, including but not limited to 1:1, 6:1, 12:1, 18:1, 24:1, 30:1, 36:1, 42:1, 48:1, 54:1 or 60:1, preferably (1-36):1, and further preferably (1-30):1.

[0057] Preferably, the solution mixing and standing time in S3 is 1 to 50 min, including but not limited to 2 min, 5 min, 8 min, 10 min, 20 min, 30 min, 40 min or 50 min, preferably 5 to 40 min, more preferably 10 to 30 min;

[0058] Preferably, the dialysis time in S4 is 0.5 to 24 hours, including but not limited to 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours or 24 hours, preferably 4 to 18 hours, and more preferably 6 to 12 hours;

[0059] Preferably, in S1, S2, S3 and S4, the mixing temperature is 10-50°C, including but not limited to 12°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or 50°C, preferably 20-50°C, further preferably 25-35°C.

[0060] As a preferred technical solution, the method for preparing hepatitis synergistic therapeutic lipid nanoparticles comprises the following steps:

[0061] (1) Weighing a certain amount of ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid and phospholipid polyethylene glycol derivative at a mass concentration of 0.1 to 75 μg / μL of the five lipid molecules in ethanol at 10 to 50° C., dissolving them in ethanol respectively, and mixing them evenly in a molar ratio of 50:(0 to 15):38.5:(0.1 to 15):1.5 to obtain an organic phase;

[0062] (2) dissolving the siRNA drug in DEPC water at a mass concentration of 0.1 to 3.0 μg / μL at 10 to 50° C., and mixing the mixture with an acidic buffer solution having a pH value less than 7.4 at a volume ratio of 1:(1 to 30) to obtain an aqueous phase;

[0063] (3) adding a certain volume of the organic phase to the aqueous phase at a mass ratio of (1-60):1 of the five lipid mixtures in the organic phase and the siRNA drug at 10-50° C., and incubating for 1-50 minutes;

[0064] (4) The mixed solution was added into a 3500Da dialysis bag at 10-50°C, and dialyzed in 1× phosphate buffer (PBS, pH=7.4) for 0.5-24 hours to obtain hepatitis therapeutic ionizable lipid nanoparticles.

[0065] The present invention also provides an application of hepatitis synergistic therapeutic ionizable lipid nanoparticles, and the application specifically comprises: synergistically treating hepatitis with the hepatitis synergistic therapeutic ionizable lipid nanoparticles and siRNA drugs.

[0066] Example 1

[0067] This example prepares an ionizable lipid nanoparticle, which includes an ionizable lipid, an auxiliary lipid, cholesterol, and a phospholipid polyethylene glycol derivative, and does not include a hepatitis therapeutic lipid as a control. The ionizable lipid is Dlin-MC3-DMA, the auxiliary lipid is DSPC, the phospholipid zwitterionic polymer derivative is DMG-PEG2000, the molar ratio of the ionizable lipid: auxiliary lipid: cholesterol: phospholipid polyethylene glycol derivative is 50:10:38.5:1.5, and the mass ratio of the lipid mixture to the siRNA drug is 12:1. The preparation method of the ionizable lipid nanoparticle comprises the following steps:

[0068] (1) Anhydrous ethanol solutions of ionizable lipid Dlin-MC3-DMA, auxiliary lipid DSPC, cholesterol, and phospholipid polyethylene glycol derivative DMG-PEG2000 were prepared at 25°C as mother solutions with concentrations of 53.16 μg / μL, 8.7 μg / μL, 15.92 μg / μL, and 5.2 μg / μL, respectively. A lipid mixed solution was prepared at a molar ratio of Dlin-MC3-DMA: DSPC: cholesterol: DMG-PEG2000 of 50:10:38.5:1.5 as the organic phase;

[0069] (2) dissolving the siRNA drug at a mass concentration of 0.5 μg / μL in DEPC water at 25° C. and mixing it with a pH 4.0 citric acid-sodium citrate buffer solution at a volume ratio of 1:3.5 to obtain an aqueous phase;

[0070] (3) adding a certain volume of the organic phase to the aqueous phase at a mass ratio of 12:1 of the lipid mixture and the siRNA drug in the organic phase at 25° C., and incubating for 10 minutes;

[0071] (4) The mixed solution was added to a 3500Da dialysis bag at 25°C and dialyzed in 1× phosphate buffer (PBS, pH=7.4) for 6 h to obtain L0-LNP@siRNA.

[0072] Example 2

[0073] This embodiment prepares a hepatitis synergistic therapeutic ionizable lipid nanoparticle, and the hepatitis synergistic therapeutic lipid nanoparticle includes ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid and phospholipid polyethylene glycol derivative. The ionizable lipid is Dlin-MC3-DMA, the auxiliary lipid is DSPC, the hepatitis therapeutic lipid is DLPC, the phospholipid zwitterionic polymer derivative is DMG-PEG2000, the molar ratio of the ionizable lipid: auxiliary lipid: cholesterol: hepatitis therapeutic lipid: phospholipid polyethylene glycol derivative is 50:8:38.5:2:1.5, and the mass ratio of the lipid mixture and the siRNA drug is 12:1. The preparation method of the hepatitis synergistic therapeutic ionizable lipid nanoparticle includes the following steps:

[0074] (1) Anhydrous ethanol solutions of ionizable lipid Dlin-MC3-DMA, auxiliary lipid DSPC, cholesterol, hepatitis therapeutic lipid DLPC and phospholipid polyethylene glycol derivative DMG-PEG2000 were prepared at 25°C as mother solutions with concentrations of 53.16 μg / μL, 8.7 μg / μL, 15.92 μg / μL, 3.4 μg / μL and 5.2 μg / μL, respectively. A lipid mixed solution was prepared according to the molar ratio of Dlin-MC3-DMA: DSPC: cholesterol: DLPC: DMG-PEG2000 of 50:8:38.5:2:1.5 as the organic phase;

[0075] (2) dissolving the siRNA drug at a mass concentration of 0.5 μg / μL in DEPC water at 25° C. and mixing it with a pH 4.0 citric acid-sodium citrate buffer solution at a volume ratio of 1:3.5 to obtain an aqueous phase;

[0076] (3) adding a certain volume of the organic phase to the aqueous phase at a mass ratio of 12:1 of the lipid mixture and the siRNA drug in the organic phase at 25° C., and incubating for 10 minutes;

[0077] (4) The mixed solution was added to a 3500Da dialysis bag at 25°C and dialyzed in 1× phosphate buffer (PBS, pH=7.4) for 6 h to obtain the L2-LNP@siRNA of the present invention.

[0078] Example 3

[0079] This embodiment prepares a hepatitis synergistic therapeutic ionizable lipid nanoparticle, and the hepatitis synergistic therapeutic lipid nanoparticle includes an ionizable lipid, an auxiliary lipid, cholesterol, a hepatitis therapeutic lipid, and a phospholipid polyethylene glycol derivative. The ionizable lipid is Dlin-MC3-DMA, the auxiliary lipid is DSPC, the hepatitis therapeutic lipid is DLPC, the phospholipid zwitterionic polymer derivative is DMG-PEG2000, the molar ratio of the ionizable lipid: auxiliary lipid: cholesterol: hepatitis therapeutic lipid: phospholipid polyethylene glycol derivative is 50:6:38.5:4:1.5, and the mass ratio of the lipid mixture to the siRNA drug is 12:1. The preparation method of the hepatitis synergistic therapeutic ionizable lipid nanoparticle includes the following steps:

[0080] (1) Anhydrous ethanol solutions of ionizable lipid Dlin-MC3-DMA, auxiliary lipid DSPC, cholesterol, hepatitis therapeutic lipid DLPC and phospholipid polyethylene glycol derivative DMG-PEG2000 were prepared at 25°C as mother solutions with concentrations of 53.16 μg / μL, 8.7 μg / μL, 15.92 μg / μL, 3.4 μg / μL and 5.2 μg / μL, respectively. A lipid mixed solution was prepared according to the molar ratio of Dlin-MC3-DMA: DSPC: cholesterol: DLPC: DMG-PEG2000 of 50:6:38.5:4:1.5 as the organic phase;

[0081] (2) dissolving the siRNA drug at a mass concentration of 0.5 μg / μL in DEPC water at 25° C. and mixing it with a pH 4.0 citric acid-sodium citrate buffer solution at a volume ratio of 1:3.5 to obtain an aqueous phase;

[0082] (3) adding a certain volume of the organic phase to the aqueous phase at a mass ratio of 12:1 of the lipid mixture and the siRNA drug in the organic phase at 25° C., and incubating for 10 minutes;

[0083] (4) The mixed solution was added to a 3500Da dialysis bag at 25°C and dialyzed in 1× phosphate buffer (PBS, pH=7.4) for 6 h to obtain the L4-LNP@siRNA of the present invention.

[0084] Example 4

[0085] This embodiment prepares a hepatitis synergistic therapeutic ionizable lipid nanoparticle, and the hepatitis synergistic therapeutic lipid nanoparticle includes ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid and phospholipid polyethylene glycol derivative. The ionizable lipid is Dlin-MC3-DMA, the auxiliary lipid is DSPC, the hepatitis therapeutic lipid is DLPC, the phospholipid zwitterionic polymer derivative is DMG-PEG2000, the molar ratio of the ionizable lipid: auxiliary lipid: cholesterol: hepatitis therapeutic lipid: phospholipid polyethylene glycol derivative is 50:4:38.5:6:1.5, and the mass ratio of the lipid mixture and the siRNA drug is 24:1. The preparation method of the hepatitis synergistic therapeutic ionizable lipid nanoparticle includes the following steps:

[0086] (1) Anhydrous ethanol solutions of ionizable lipid Dlin-MC3-DMA, auxiliary lipid DSPC, cholesterol, hepatitis therapeutic lipid DLPC and phospholipid polyethylene glycol derivative DMG-PEG2000 were prepared at 25°C as mother solutions with concentrations of 53.16 μg / μL, 8.7 μg / μL, 15.92 μg / μL, 3.4 μg / μL and 5.2 μg / μL, respectively. A lipid mixed solution was prepared according to the molar ratio of Dlin-MC3-DMA: DSPC: cholesterol: DLPC: DMG-PEG2000 of 50:4:38.5:6:1.5 as the organic phase;

[0087] (2) dissolving the siRNA drug at a mass concentration of 0.5 μg / μL in DEPC water at 25° C. and mixing it with a pH 4.0 citric acid-sodium citrate buffer solution at a volume ratio of 1:3.5 to obtain an aqueous phase;

[0088] (3) adding a certain volume of the organic phase to the aqueous phase at a mass ratio of 24:1 of the lipid mixture and the siRNA drug in the organic phase at 25° C., and incubating for 10 minutes;

[0089] (4) The mixed solution was added to a 3500Da dialysis bag at 25°C and dialyzed in 1× phosphate buffer (PBS, pH=7.4) for 6 h to obtain the L6-LNP@siRNA of the present invention.

[0090] Example 5

[0091] This embodiment prepares a hepatitis synergistic therapeutic ionizable lipid nanoparticle, and the hepatitis synergistic therapeutic lipid nanoparticle includes an ionizable lipid, an auxiliary lipid, cholesterol, a hepatitis therapeutic lipid, and a phospholipid polyethylene glycol derivative. The ionizable lipid is Dlin-MC3-DMA, the auxiliary lipid is DSPC, the hepatitis therapeutic lipid is DLPC, the phospholipid zwitterionic polymer derivative is DMG-PEG2000, the molar ratio of the ionizable lipid: auxiliary lipid: cholesterol: hepatitis therapeutic lipid: phospholipid polyethylene glycol derivative is 50:2:38.5:8:1.5, and the mass ratio of the lipid mixture to the siRNA drug is 12:1. The preparation method of the hepatitis synergistic therapeutic ionizable lipid nanoparticle includes the following steps:

[0092] (1) Anhydrous ethanol solutions of ionizable lipid Dlin-MC3-DMA, auxiliary lipid DSPC, cholesterol, hepatitis therapeutic lipid DLPC and phospholipid polyethylene glycol derivative DMG-PEG2000 were prepared at 25°C as mother solutions with concentrations of 53.16 μg / μL, 8.7 μg / μL, 15.92 μg / μL, 3.4 μg / μL and 5.2 μg / μL, respectively. A lipid mixed solution was prepared according to the molar ratio of Dlin-MC3-DMA: DSPC: cholesterol: DLPC: DMG-PEG2000 of 50:2:38.5:4:1.5 as the organic phase;

[0093] (2) dissolving the siRNA drug at a mass concentration of 0.5 μg / μL in DEPC water at 25° C. and mixing with a pH 4.0 citric acid-sodium citrate buffer solution at a volume ratio of 1:3.5 to obtain an aqueous phase;

[0094] (3) adding a certain volume of the organic phase to the aqueous phase at a mass ratio of 12:1 of the lipid mixture and the siRNA drug in the organic phase at 25° C., and incubating for 30 minutes;

[0095] (4) The mixed solution was added to a 3500Da dialysis bag at 25°C and dialyzed in 1× phosphate buffer (PBS, pH=7.4) for 6 h to obtain the L8-LNP@siRNA of the present invention.

[0096] Example 6

[0097] This embodiment prepares a hepatitis synergistic therapeutic ionizable lipid nanoparticle, and the hepatitis synergistic therapeutic lipid nanoparticle includes ionizable lipid, cholesterol, hepatitis therapeutic lipid and phospholipid polyethylene glycol derivative. The ionizable lipid is Dlin-MC3-DMA, the hepatitis therapeutic lipid is DLPC, the phospholipid zwitterionic polymer derivative is DMG-PEG2000, the molar ratio of the ionizable lipid: cholesterol: hepatitis therapeutic lipid: phospholipid polyethylene glycol derivative is 50:38.5:10:1.5, and the mass ratio of the lipid mixture to the siRNA drug is 12:1. The preparation method of the hepatitis synergistic therapeutic ionizable lipid nanoparticle includes the following steps:

[0098] (1) Anhydrous ethanol solutions of ionizable lipid Dlin-MC3-DMA, cholesterol, hepatitis therapeutic lipid DLPC, and phospholipid polyethylene glycol derivative DMG-PEG2000 were prepared at 25°C as mother solutions with concentrations of 53.16 μg / μL, 15.92 μg / μL, 3.4 μg / μL, and 5.2 μg / μL, respectively. A lipid mixed solution was prepared at a molar ratio of Dlin-MC3-DMA: cholesterol: DLPC: DMG-PEG2000 of 50:38.5:10:1.5 as the organic phase;

[0099] (2) dissolving the siRNA drug at a mass concentration of 0.5 μg / μL in DEPC water at 25° C. and mixing it with a pH 4.0 citric acid-sodium citrate buffer solution at a volume ratio of 1:3.5 to obtain an aqueous phase;

[0100] (3) adding a certain volume of the organic phase to the aqueous phase at a mass ratio of 12:1 of the lipid mixture and the siRNA drug in the organic phase at 25° C., and incubating for 10 minutes;

[0101] (4) The mixed solution was added to a 3500Da dialysis bag at 25°C and dialyzed in 1× phosphate buffer (PBS, pH=7.4) for 18 hours to obtain the L10-LNP@siRNA of the present invention.

[0102] Experimental Example 1 Detection of the particle size of ionizable lipid nanoparticles for synergistic therapeutic effects on hepatitis

[0103] The size of insulin nanoparticles was measured using a Nano-ZS90 laser particle size analyzer. The refractive index was set to 1.590, the absorption coefficient was set to 0.010, the temperature was set to 25°C, the measurement mode was set to automatic, and the Z average statistical value was used as the measurement result. Figure 1 As shown, the particle sizes of the hepatitis synergistic therapeutic ionizable lipid nanoparticles prepared in Examples 2 and 3 are all less than 200 nm.

[0104] Experimental Example 2 Detection of siRNA Drug Encapsulation Efficiency in Hepatitis Co-therapeutic Ionizable Lipid Nanoparticles

[0105] Quant-iT TM RNA method: dilute the concentrated buffer (component B) 20 times with DEPC water to make 1×Te working solution. TM The RNA reagent was melted at room temperature, and then the concentrated DMSO stock solution (component A) was diluted 200 times with 1×Te solution. The concentration of RNA standard (component C) was 100ug / mL, and it was diluted 50 times with 1×Te working solution to make a 2ug / mL working solution. The hepatitis therapeutic ionizable lipid nanoparticles were diluted to 100uL with PBS, and 100uL component A was added. Fluorescence test was performed at an excitation wavelength of 480nm and an emission wavelength of 520nm. The embedding rate was calculated according to formula (1). The experimental results are shown in Figure 2 As shown, the encapsulation rates of the hepatitis synergistic therapeutic ionizable lipid nanoparticles prepared in Examples 2 and 3 were 93.15±1.50% and 90.02±2.55%, respectively.

[0106] Embedding rate (%) = (m-m 1 ) / m ×100%(1)

[0107] Among them, m 1 is the mass of free siRNA; m is the total mass of siRNA input during particle preparation.

[0108] Experimental Example 3 Study on Cytotoxicity

[0109] This experimental example studies the cytotoxicity of hepatitis synergistic therapeutic ionizable lipid nanoparticles loaded with siRNA drugs.

[0110] The toxicity of auxiliary lipids and hepatitis therapeutic lipid nanoparticles containing different molar ratios on RAW 264.7 cells was detected by MTT assay. 1.0×10 4 RAW 264.7 cells were added with 100 μL of DMEM medium containing 10% fetal bovine serum and 1% double antibody. After culturing in the incubator for 24 hours, the medium was removed and hepatitis therapeutic ionizable lipid nanoparticles with a siRNA concentration of 100 nM were added. After culturing for 24 hours, 20 μL of MTT (5 mg / mL dissolved in PBS) was added to each well and the culture was continued for 4 hours. MTT was removed and 150 μL of dimethyl sulfoxide (DMSO) was added to each well and incubated on a shaker at 37°C for 10 minutes. The microplate reader was preheated in advance, and the detection was performed at a wavelength of 490 nm, and the cell survival rate was calculated. The experimental results are shown in the figure. Figure 3 As shown, the hepatitis synergistic therapeutic ionizable lipid nanoparticles prepared in Examples 1, 2 and 3 have no obvious cytotoxicity, demonstrating that they have good biocompatibility.

[0111] Experimental Example 4: Animal-level targeting study of ionizable lipid nanoparticles for synergistic treatment of hepatitis

[0112] This test example studies the animal level biodistribution of the ionizable lipid nanoparticles of Examples 1, 2 and 3. Female Balb / c mice were randomly divided into 4 groups and injected with PBS or Cy5-siRNA@LNP (0.35 mg / kg) via the tail vein. The animals in each group were euthanized and dissected 6 hours after the injection. The Cy5 fluorescence signal distribution in the heart, lung, liver, spleen, kidney and other tissues was examined using the PerkinElmer IVIS Spectrum in vivo imaging system. Figure 4 As shown, the ionizable lipid nanoparticles prepared in Examples 1 and 2 have a good liver targeting effect.

[0113] Experimental Example 5 Study on the therapeutic effect of synergistic therapeutic ionizable lipid nanoparticles on hepatitis at the animal level

[0114] In this test example, the therapeutic effects of Examples 1 and 2 on hepatitis mice stimulated with lipopolysaccharide (LPS) and D-galactosamine (D-GaIN) were studied.

[0115] Female Balb / c mice weighing 18-25g were divided into 6 groups and injected with PBS, siRNA, L2-LNP@Scr siRNA, L2-LNP@TNF-α siRNA and L0-LNP@TNF-α siRNA through the tail vein. LPS (100μg / kg) and D-GaIN (1.25g / kg) were injected intraperitoneally 24h later. Blood samples were collected and dissected 6h after stimulation. Mice in the control group were not stimulated and injected with an equal volume of PBS. Blood samples were collected and samples were collected 24h later.

[0116] After LPS and D-GaIN stimulation, the experimental results are as follows Figure 5 As shown in the figure, the TNF-α content in the serum of mice in the PBS group increased, and the TNF-α content in the serum of mice treated with L2-LNP@Scr siRNA in advance was lower than that in the stimulation group, indicating that DLPC can inhibit the expression of inflammatory factors, and the TNF-α content in the serum of mice treated with L2-LNP@TNF-αsiRNA was significantly reduced, and compared with L0-LNP@TNF-αsiRNA, the ability to inhibit the expression of inflammatory factors is stronger. Figure 6 As shown, analysis of biomarkers related to liver function found that L2-LNP@Scr siRNA could reduce the level of liver function markers, but L2-LNP@TNF-α siRNA could make the marker level reach the level of the control group, proving that the combination of L2-LNP and TNF-α siRNA can exert a more superior therapeutic effect and enable better treatment of hepatitis diseases.

[0117] The above is a detailed introduction to a hepatitis synergistic therapeutic ionizable lipid nanoparticle and its preparation method and application provided in the examples of the present application. The description of the above embodiments is only used to help understand the method and its core idea of ​​the present application; at the same time, for those of ordinary skill in the art, according to the idea of ​​the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

[0118] For example, certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that hardware manufacturers may use different nouns to refer to the same component. This specification and claims do not use differences in names as a way to distinguish components, but use differences in the functions of components as the criteria for distinction. As mentioned throughout the specification and claims, "including" and "comprising" are open-ended terms, so they should be interpreted as "including / including but not limited to". "Approximately" means that within an acceptable error range, those skilled in the art can solve the technical problem within a certain error range and basically achieve the technical effect. The subsequent description of the specification is a preferred embodiment of the present application, but the description is for the purpose of illustrating the general principles of the present application, and is not used to limit the scope of the present application. The scope of protection of the present application shall be determined by the definition of the attached claims.

[0119] It should also be noted that the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a product or system including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such a product or system. In the absence of more restrictions, the elements defined by the sentence "comprises a ..." do not exclude the existence of other identical elements in the product or system including the elements.

[0120] It should be understood that the term "and / or" used in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0121] The above description shows and describes several preferred embodiments of the present application, but as mentioned above, it should be understood that the present application is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the application concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not deviate from the spirit and scope of the present application, and should be within the scope of protection of the claims attached to the present application.

Claims

1. A hepatitis synergistic therapeutic ionizable lipid nanoparticle, characterized in that: The hepatitis synergistic therapeutic ionizable lipid nanoparticles comprise ionizable lipids, auxiliary lipids, cholesterol, hepatitis therapeutic lipids, phospholipid polyethylene glycol derivatives and siRNA drugs.

2. The hepatitis synergistic therapeutic ionizable lipid nanoparticles according to claim 1, characterized in that: The ionizable lipids include one or more of 4-(N,N-dimethylamino)butyric acid (dialene) methyl ester, 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecanyloxy)hexyl]amino]-octanoate and ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

3. The hepatitis synergistic therapeutic ionizable lipid nanoparticles according to claim 1, characterized in that: The auxiliary lipids include one or more of 1,2-distearoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 2-oleoyl-1-palmitoyltinglycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dierucoyl-sn-glycero-3-phosphocholine.

4. The hepatitis synergistic therapeutic ionizable lipid nanoparticles according to claim 1, characterized in that: The hepatitis therapeutic lipid is 1,2-dilauroyl-sn-glycero-3-phosphocholine.

5. The hepatitis synergistic therapeutic ionizable lipid nanoparticles according to claim 1, characterized in that: The phospholipid polyethylene glycol derivative is 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000.

6. A method for preparing hepatitis synergistic therapeutic ionizable lipid nanoparticles, used for preparing the hepatitis synergistic therapeutic ionizable lipid nanoparticles according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: S1: Weigh a certain mass of ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid and phospholipid polyethylene glycol derivative respectively, dissolve them in ethanol respectively, mix them evenly in a certain molar ratio, and obtain an organic phase; S2: dissolving the siRNA drug in DEPC water at a certain concentration, and mixing it with the acidic buffer solution at a certain volume ratio to obtain an aqueous phase; S3: adding a certain volume of the organic phase to the aqueous phase according to a certain mass ratio of the lipid mixture and the siRNA drug in the organic phase, mixing, and incubating the mixture to obtain a mixed solution; S4: The mixed solution is added into a 3500Da dialysis bag and dialyzed in 1× phosphate buffer for a certain period of time to obtain hepatitis synergistic therapeutic ionizable lipid nanoparticles.

7. The preparation method according to claim 6, characterized in that: The molar ratio of the ionizable lipid, auxiliary lipid, cholesterol, hepatitis therapeutic lipid and phospholipid polyethylene glycol derivative in the S1 is 50:(0-15):38.5:(0.1-15):1.

5.

8. The preparation method according to claim 6, characterized in that: The mass concentration of the siRNA aqueous solution in S2 is 0.1-3.0 μg / μL, the pH of the acidic buffer solution is less than 7.4, and the volume ratio of the siRNA aqueous solution to the acidic buffer solution is 1:(1-30).

9. The preparation method according to claim 6, characterized in that: The mass ratio of the five lipid mixtures in S3 to the siRNA drug is (1-60):

1.

10. An application of ionizable lipid nanoparticles for synergistic treatment of hepatitis, characterized in that: The specific application is: using the hepatitis synergistic therapeutic ionizable lipid nanoparticles according to any one of claims 1 to 5 in conjunction with siRNA drugs to synergistically treat hepatitis.

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