Lipid nano-particles for improving liver targeting of medicine as well as preparation method and application of lipid nano-particles

Abstract

The invention specifically discloses lipid nanoparticles for improving liver targeting of a drug as well as a preparation method and application of the lipid nanoparticles, and relates to the technical field of biological medicines. The invention provides lipid nanoparticles for intramuscular injection, the lipid nanoparticles comprise PC phospholipid and an active component, the active component is a nucleic acid drug, and the lipid nanoparticles can improve liver expression or distribution of the active component. The invention provides a theoretical basis and an experimental basis for developing a new treatment strategy, and has good clinical transformation and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to liver-targeted lipid nanoparticles and their preparation methods and applications. Background Art

[0002] Tyrosinemia is a rare autosomal recessive genetic metabolic disease. Due to the disorder of tyrosine degradation, it causes damage to multiple organs such as the brain, liver, kidney, and bones, with poor prognosis and high lethality and disability rates. Currently, the traditional treatment strategies for tyrosinemia mainly include dietary control and liver transplantation. However, these methods have problems such as unstable control and limited liver sources, and cannot meet the clinical needs. In recent years, the rapid development of mRNA gene therapy technology has provided a new treatment strategy for treating tyrosinemia. The mRNA protein replacement therapy can express the target protein in the liver, thereby treating genetic diseases caused by protein deficiency.

[0003] Currently, there is an international product that encapsulates mRNA in lipid nanoparticles (LNP) and targets and delivers mRNA to the liver through intravenous administration for the treatment of rare liver diseases. However, preclinical and clinical studies have shown that mRNA / LNP for protein replacement therapy still faces problems such as strong inflammatory reactions of LNP and anti-vector antibodies, which limit the further application of LNP in mRNA protein replacement therapy. In addition, traditional intravenous injection has problems such as intravenous injection reactions, long administration time, and poor patient compliance. The method of local administration instead of intravenous administration has the characteristics of convenient administration, short administration time, and high patient compliance, and has been used in preclinical studies of mRNA protein replacement therapy for treating rare liver diseases by mRNA biotechnology companies. However, only about 20% of the mRNA is distributed in the liver after intramuscular administration of LNP used clinically, resulting in low liver distribution ratio and insufficient protein expression after intramuscular administration. Summary of the Invention

[0004] To solve the above problems, the present invention provides a lipid nanoparticle for intramuscular injection, characterized in that the lipid nanoparticle contains PC phospholipids and an active ingredient, the active ingredient is a nucleic acid drug, and the lipid nanoparticle can improve the liver expression or distribution of the active ingredient.

[0005] In one embodiment, the lipid nanoparticle further contains PEG lipid.

[0006] In one embodiment, the lipid nanoparticle further contains structural lipid.

[0007] In one embodiment, the lipid nanoparticle further contains ionizable lipid.

[0008] In one embodiment, the ionizable lipid comprises one or a combination of DLin-MC3-DMA (MC3), ALC-0315, and SM-102.

[0009] In one embodiment, the ionizable lipid is ALC-0315.

[0010] Phospholipids are used to encapsulate and protect the core formed by the interaction of ionizable lipids and drugs within the lipid nanoparticles and bind to the phospholipid bilayer of the target cells to facilitate cell membrane penetration and endosomal escape during the intracellular delivery of the drugs.

[0011] In one embodiment, the PC phospholipids include one or a combination of distearoyl phosphatidylcholine (DSP C), palmitoyl oleoyl phosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-docosanoyl-sn-glycero-phosphocholine (DUPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC).

[0012] In one embodiment, the PC phospholipids include one or a combination of DMPE, DPPE, DSPE, DOP E, DMPC, DPPC, DSPC, DOPC.

[0013] In one embodiment, the PC phospholipid is DMPC.

[0014] Structural lipids are used to impart rigidity to the lipid load within the lipid nanoparticles in terms of morphology and improve the stability of the nanoparticles by dispersing in the core and surface of the nanoparticles.

[0015] In one embodiment, the structural lipids include one or a combination of cholesterol, cholestenol, spinasterol, coprostanol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and α-tocopherol.

[0016] In one embodiment, the structural lipid is cholesterol.

[0017] PEG-lipids contribute to the particle stability of the nanoparticles in serum within the lipid nanoparticles and act as a barrier to nanoparticle aggregation.

[0018] In one embodiment, the PEG lipid includes one or a combination of ALC-0159, PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

[0019] In one embodiment, the PEG lipid is PEG-DMG.

[0020] In one embodiment, the molar ratio of the ionizable lipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 20-50.

[0021] In one embodiment, the molar ratio of the ionizable lipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 30-40.

[0022] In one embodiment, the molar ratio of the ionizable lipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 30.

[0023] In one embodiment, the molar ratio of the PC phospholipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 5-25.

[0024] In one embodiment, the molar ratio of the PC phospholipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 5-15.

[0025] In one embodiment, the molar ratio of the PC phospholipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 15.

[0026] In one embodiment, the molar ratio of the structural lipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 25-60.

[0027] In one embodiment, the molar ratio of the structural lipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 35-60.

[0028] In one embodiment, the molar ratio of the structural lipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 53.5.

[0029] In one embodiment, the molar ratio of the PEG lipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 1-2.5.

[0030] In one embodiment, the molar ratio of the PEG lipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 1.5-2.

[0031] In one embodiment, the molar ratio of PEG lipid in the lipid nanoparticles to the lipid component in the lipid nanoparticles is 1.5.

[0032] In one embodiment, the nucleic acid includes one or a combination of siRNA, rRNA, DNA, aptamer, mRNA, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, DNA enzyme.

[0033] In one embodiment, the nucleic acid is mRNA.

[0034] In one embodiment, the mRNA includes one or a combination of mRNA-3927 and mRNA-3705.

[0035] In one embodiment, the mRNA is mRNA-3927.

[0036] In one embodiment, the lipid nanoparticles include: ALC-0315, DMPC, cholesterol, PEG-DMG, mRNA-3927.

[0037] The present invention also provides, in another aspect, the use of a pharmaceutical composition containing the above lipid nanoparticles in the preparation of a drug for preventing and / or treating liver diseases.

[0038] In one embodiment, the liver diseases include hereditary metabolic rare liver diseases.

[0039] In one embodiment, the hereditary metabolic rare liver diseases include one or a combination of propionic academia, methylmalonic academia, ornithine carbamoyltransferase deficiency, and tyrosinemia.

[0040] In one embodiment, the lipid nanoparticles can be used in combination with an agent.

[0041] In one embodiment, the agent includes one or a combination of an anti-inflammatory compound, a steroid (such as a corticosteroid), a statin, estradiol, a BTK inhibitor, an S1P1 agonist, a glucocorticoid receptor modulator (GRM), or an antihistamine.

[0042] In one embodiment, the LNP can be used in combination with dexamethasone, methotrexate, acetaminophen, an H1 receptor blocker, or an H2 receptor blocker.

[0043] In one embodiment, the subject in need is pre-treated with one or more agents before administering the above lipid nanoparticles. In some embodiments, the subject can be pre-treated with dexamethasone, methotrexate, acetaminophen, an H1 receptor blocker, or an H2 receptor blocker in a suitable amount (e.g., 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or any other suitable amount). The pre-treatment can occur 24 hours or less before the administration of the lipid nanoparticles (e.g., 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes) and can occur one, two, or more times, for example, at increasing doses.

[0044] In one embodiment, the lipid nanoparticles are administered by intranasal (e.g., by inhalation), intrathecal (into the intraspinal canal or subarachnoid space), intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, ocular, sublingual, oral (by ingestion), intracerebral, and transdermal (by absorption, e.g., through a skin catheter) administration methods.

[0045] In one embodiment, the lipid nanoparticles are administered by intramuscular administration method.

[0046] In another aspect, the present invention also provides a pharmaceutical composition comprising the above lipid nanoparticles.

[0047] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically or immunologically acceptable carrier or excipient.

[0048] In another aspect, the present invention also provides a pharmaceutical preparation comprising the above pharmaceutical composition.

[0049] In another aspect, the present invention also provides a lipid nanoparticle, a drug, a pharmaceutical composition, or a pharmaceutical preparation administered by intramuscular injection.

[0050] In another aspect, the present invention also provides the use of the above lipid nanoparticles, drugs, pharmaceutical compositions, or pharmaceutical preparations in the preparation of drugs for preventing and / or treating liver diseases.

[0051] Compared with the prior art, the beneficial effects of the present invention are:

[0052] 1. Significantly improve the expression intensity of mRNA in the liver after intramuscular administration.

[0053] 2. Significantly improve the expression distribution ratio of mRNA in the liver / muscle after intramuscular administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0055] Figure 1 is the influence of the components of lipid nanoparticles on the liver targeting of drugs;

[0056] Figure 2 is the influence of the molar ratio of the components of lipid nanoparticles on the liver targeting of drugs.

[0057] Figure 3 is the influence of the combination of lipid nanoparticles and dexamethasone on the liver targeting of drugs. Detailed Embodiments

[0058] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0059] As used herein, "comprising", "having", or "including" includes "containing", "consisting essentially of", "consisting substantially of", and "consisting of"; "consisting essentially of", "consisting substantially of", and "consisting of" are sub-concepts of "comprising", "having", or "including".

[0060] Definitions

[0061] As used herein, the term "liver disease" refers to diseases occurring in the liver, which may include metabolic liver diseases (simple steatosis, non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), propionic acidemia, methylmalonic acidemia, tyrosinemia, ornithine carbamoyltransferase deficiency), liver inflammation, and other congenital and acquired liver injuries or dysfunctions caused by different reasons (cholestatic liver diseases, liver fibrosis, cirrhosis, hepatic decompensation, liver cancer).

[0062] As used herein, the term "delivery" refers to providing an entity to a destination. In some embodiments, delivering a therapeutic and / or prophylactic agent to a subject can involve administering to the subject an LNP comprising the therapeutic and / or prophylactic agent (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering an LNP to a mammal or mammalian cell can involve contacting one or more cells with the lipid nanoparticle.

[0063] As used herein, the term "enhanced hepatic drug targeting" means that, compared to the level of delivery of a therapeutic and / or prophylactic agent (e.g., Luciferase mRNA) to a target tissue by a control nanoparticle, a nanoparticle delivers more (e.g., at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) therapeutic and / or prophylactic agent to a target tissue (e.g., the mammalian liver). The level of delivery of a nanoparticle to a particular tissue can be measured by comparing the amount of protein produced in the tissue to the weight of the tissue, comparing the amount of therapeutic and / or prophylactic agent in the tissue to the weight of the tissue, comparing the amount of protein produced in the tissue to the total protein in the tissue, or comparing the amount of therapeutic and / or prophylactic agent in the tissue to the total therapeutic and / or prophylactic agent in the tissue. It should be understood that enhanced hepatic drug targeting by a nanoparticle need not be determined in the subject being treated and can be determined in an alternative such as an animal model (e.g., a rat model).

[0064] As used herein, "expression" of a nucleic acid refers to the translation of mRNA into a polypeptide or protein and / or post-translational modification of the polypeptide or protein.

[0065] As used herein, "route of administration" can include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. A route of administration can be selected to target delivery (e.g., specifically deliver) to a particular region or system of the body.

[0066] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that comprises a polyethylene glycol moiety.

[0067] As used herein, "polymeric lipid" refers to a lipid that contains repeating subunits in its chemical structure. In some embodiments, the polymeric lipid is a lipid that comprises a polymeric moiety. In some embodiments, the polymeric lipid is a PEG lipid. In some embodiments, the polymeric lipid is not a PEG lipid. In some embodiments, the polymeric lipid is Brij or OH-PEG-stearate.

[0068] As used herein, "phospholipid" is a lipid that includes a phosphate ester moiety and one or more carbon chains (such as unsaturated fatty acid chains). The phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more degrees of unsaturation). The phospholipid or its analogs or derivatives may include choline. The phospholipid or its analogs or derivatives may not include choline. Certain phospholipids may promote fusion with membranes. In some embodiments, cationic phospholipids may interact with one or more negatively charged phospholipids of a membrane (e.g., a cell or an intracellular membrane). Fusion of the phospholipid with the membrane may allow one or more elements of a lipid-containing composition to be delivered through the membrane, thereby allowing, for example, delivery of the one or more elements to a cell.

[0069] As used herein, "polydispersity index" and "PDI" are used interchangeably and are a ratio that describes the homogeneity of the particle size distribution of a system. For example, a small value less than 0.3 indicates a narrow particle size distribution.

[0070] As used herein, "RNA" refers to ribonucleic acid, which may be naturally or non-naturally occurring. In some embodiments, the RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. The RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. The RNA may have a nucleotide sequence encoding a target polypeptide. In some embodiments, the RNA may be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide (e.g., in vivo translation of an mRNA inside a mammalian cell) may produce the encoded polypeptide. The RNA is optionally selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, long non-coding RNA (lncRNA), and mixtures thereof.

[0071] mRNA-3927

[0072] As used herein, the term "mRNA-3927" refers to mRNA-3927 for the treatment of propionic acidemia as disclosed in patent WO2023287751A1. The specific sequence of the mRNA-3927 is disclosed in patent WO2023287751A1, which is incorporated herein by reference in its entirety.

[0073] mRNA-3705

[0074] As used herein, the term "mRNA-3705" refers to mRNA-3705 for the treatment of methylmalonic acidemia as disclosed in Patent US20240269248A1. The specific sequence of said mRNA-3705 is disclosed in Patent US20240269248A1, which is incorporated herein by reference in its entirety.

[0075] As used herein, the term "subject" refers to any organism to which a composition or formulation according to the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0076] As used herein, the term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0077] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is a carrier for the administration of therapeutic agents, including various excipients and diluents. The term refers to those pharmaceutical carriers that are not necessarily the active ingredient themselves and that have no excessive toxicity upon administration. Suitable carriers are well known to those of ordinary skill in the art, and a full discussion of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences, Mack Pub. Co., N.J. 1991.

[0078] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH regulators, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. that are compatible with the administration of a drug. The use of such media and agents for a pharmaceutically active substance is well known in the art. The composition may contain other active compounds that provide supplementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin.

[0079] The pharmaceutical compositions disclosed in the present invention can be in the form of granules, tablets, lyophilized powders, suppositories, capsules, sublingual tablets, liquid solutions, nasal drops, sprays, metered spray forms.

[0080] Any known method can be used to administer the pharmaceutical composition of the present invention. The terms "administer" or "administration" of a substance, compound, or agent to a subject can be effected by one of a variety of methods known to those skilled in the art.

[0081] For example, the compound or agent can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ophthalmically, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). The compound or reagent can also be suitably introduced by a rechargeable or biodegradable polymeric device or other device (e.g., patches and pumps or formulations) that provides for extended, slow, or controlled release of the compound, reagent. Administration can also be effected, for example, once, multiple times, and / or over one or more extended periods.

[0082] The administration routes can include: (1) direct naked DNA or protein injection method; (2) linking the cDNA, mRNA, and protein of the protein with a transferrin / poly-L-lysine complex to enhance its biological effect; (3) forming a complex of cDNA, mRNA, and protein with a positively charged lipid to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) mediating the entry of cDNA, mRNA, and protein into cells after encapsulation with liposomes, which is not only conducive to the smooth entry of macromolecules but also protected from hydrolysis by various extracellular enzymes; (5) binding cDNA, mRNA, and protein with cholesterol to increase the cytoplasmic retention time by 10 times; (6) transporting cDNA, mRNA, and protein with immunoliposomes can specifically transport them to target tissues and target cells; (7) in vitro transfection of cDNA, mRNA, and protein to transfected cells (such as fibroblasts) can also better load protein-related drugs into target cells; (8) electroporation, that is, introducing cDNA, mRNA, and protein into target cells with the aid of an electric current.

[0083] As used herein, the term "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level includes factors such as the type and severity of the subject, age, sex, drug activity, sensitivity to the drug, administration time, administration route, and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.

[0084] As used herein, the term "treating" a condition or a patient refers to taking steps to achieve a beneficial or desired result, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviating, substantially inhibiting, slowing down or reversing the progression of a disease, condition or disorder, substantially improving or alleviating the clinical or aesthetic symptoms of a condition, substantially preventing the clinical or aesthetic symptoms of a disease, condition or disorder, and avoiding adverse or annoying symptoms. Treatment also refers to accomplishing one or more of the following: (a) reducing the severity of a disorder; (b) limiting the development of characteristic symptoms of the disorder being treated; (c) limiting the worsening of characteristic symptoms of the disorder being treated; (d) limiting the recurrence of the disorder in patients previously suffering from the disorder; and / or (e) limiting the recurrence of symptoms in patients previously without symptoms of the disorder.

[0085] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0086] Example 1

[0087] Effect of lipid nanoparticle components on the liver targeting of drugs:

[0088] 1. Preparation of solutions:

[0089] (1) Preparation of lipid mixture: According to the prescription table in Table 1, the co-lipids (DMPE, DPPE, DSPE, DOPE, DMPC, DPPC, DSPC, DOPC) and PEG-lipids (ALC-0159 and mPEG-DMG) were respectively configured into lipid solutions according to the molar ratio of ionizable lipid / co-lipid / cholesterol / PEG-lipid of 50 / 10 / 38.5 / 1.5, and the total lipid concentration was 10 mg / ml.

[0090] (2) Preparation of mRNA working solution: Take 20 μl of Luciferase mRNA stock solution (1 mg / ml), add 380 μl of (10 mM, pH 4.0) citric acid buffer and mix well to obtain a 50 μg / ml mRNA solution.

[0091] 2. Preparation of luciferase mRNA / LNP:

[0092] (1) Using a microfluidic device (INano L+, Mai'anna), set the parameters according to the following method to prepare luciferase mRNA / LNP samples (total volume, 1.6 ml; flow rate ratio, lipid phase: aqueous phase = 1:3; flow rate, 20 ml / min). Immediately after the sample preparation, dilute 7-fold with 1X PBS for subsequent ultrafiltration.

[0093] (2) Ultrafiltration: Transfer the sample diluted 7 times as described above into a 100 kd ultrafiltration tube and perform ultrafiltration on a low-temperature centrifuge. The centrifuge settings are as follows: rotation speed: 3000 RPM, temperature: 4 °C, time: 30 min. Take out the sample when it is ultrafiltered to about 1 ml, then add 5 ml of 1X PBS and perform ultrafiltration again. Stop ultrafiltration when it is ultrafiltered to about 1 ml again to obtain the luciferase mRNA / LNP.

[0094] 3. Data Characterization

[0095] (1) Particle size and PDI detection: Take 20 μl of the sample, dilute it 50 times with ultrapure water, and perform detection on a Malvern ZETA particle size R PRO. The results are shown in Prescription Table 1.

[0096] (2) Encapsulation efficiency detection: Use a Ribogreen kit and a microplate reader for detection. The results are shown in Prescription Table 1.

[0097] (3) In vivo protein expression: For in vivo imaging of mice, take 10 μg of the above-mentioned luciferase mRNA / LNP, perform intramuscular injection on Balb / c (6 - 8 weeks) mice, inject the luciferin substrate at 3, 6, and 24 hours after administration, and place it in a small animal in vivo imager for luciferase expression imaging detection. The results are as Figure 1 shown. The luciferase mRNA / LNP with a lipid composition of ALC-0315 / DMPC / cholesterol / mPEG-DMG has the highest expression intensity in the liver, and the expression distribution ratio of liver / muscle is relatively large.

[0098] Prescription Table 1

[0099]

[0100] Example 2

[0101] Effect of molar ratio of lipid nanoparticle components on liver targeting of drugs:

[0102] Subsequently, further screen the respective prescription ratios on the basis of the ALC-0315 / DMPC / cholesterol / mPEG-DMG combination, and prepare luciferase mRNA / LNP samples according to Table 2. The preparation method and data characterization method of luciferase mRNA / LNP are the same as those in Example 1.

[0103] The particle size and PDI detection results are shown in Table 2.

[0104] The encapsulation efficiency detection results are shown in Table 2.

[0105] The in vivo protein expression detection results are asFigure 2 As shown, the luciferase mRNA / LNP with a molar ratio of ALC-0315 / DMPC / cholesterol / mPEG-DMG of 30 / 15 / 53.5 / 1.5 has a relatively high expression intensity in the liver, and the expression distribution ratio of liver / muscle is the largest.

[0106] Table 2

[0107]

[0108]

[0109] Example 3

[0110] Effect of combination of lipid nanoparticles and dexamethasone on drug liver targeting:

[0111] One hour before intramuscular administration of luciferase mRNA / LNP with a molar ratio of ALC-0315 / DMPC / cholesterol / mPEG-DMG of 30 / 15 / 53.5 / 1.5, different doses of dexamethasone were intraperitoneally injected, and the luciferin substrate was injected at 3, 6, and 24 hours after intramuscular administration, and then the animals were placed in a small animal in vivo imaging system for luciferase expression imaging detection.

[0112] The results of in vivo protein expression detection are as Figure 3 shown: Intraperitoneal injection of 2 mg / kg of dexamethasone 1 h before intramuscular administration of luciferase mRNA / LNP (molar ratio of ALC-0315 / DMPC / cholesterol / mPEG-DMG of 30 / 15 / 53.5 / 1.5) can significantly increase the expression intensity of luciferase mRNA / LNP in the liver and the expression distribution ratio of liver / muscle.

[0113] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent in such process, method, article or device. Without further limitation, an element defined by the statement "including a..." does not exclude the existence of another identical element in the process, method, article or device including the said element.

[0114] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid nanoparticle for intramuscular injection, characterized in that: The lipid nanoparticles contain PC phospholipids and active ingredients, wherein the active ingredients are nucleic acid drugs, and the lipid nanoparticles can improve the liver expression or distribution of the active ingredients.

2. The lipid nanoparticle according to claim 1, characterized in that The lipid nanoparticles further comprise PEG lipids.

3. The lipid nanoparticle according to claim 2, characterized in that The lipid nanoparticles further comprise structured lipids and / or ionizable lipids.

4. The lipid nanoparticle according to claim 3, characterized in that The molar ratio of the ionizable lipid in the lipid nanoparticle to the lipid components in the lipid nanoparticle is 20-50; and / or the molar ratio of PC phospholipids in the lipid nanoparticles to lipid components in the lipid nanoparticles is 5-25; and / or the molar ratio of the structural lipids in the lipid nanoparticles to the lipid components in the lipid nanoparticles is 25-60; And / or the molar ratio of PEG lipid in the lipid nanoparticles to the lipid components in the lipid nanoparticles is 1-2.

5.

5. The lipid nanoparticle according to claim 4, characterized in that The PC phospholipids include distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (PO PC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-docosaoyl-sn-glycero-phosphocholine (DUPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteroyl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC) or a combination thereof.

6. The lipid nanoparticle according to claim 4, characterized in that The PEG lipid includes one or a combination of ALC-0159, PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE.

7. The lipid nanoparticle according to claim 4, characterized in that The ionizable lipids include one or a combination of DLin-MC3-DMA (MC3), ALC-0315 and SM-102.

8. The lipid nanoparticle according to claim 4, characterized in that The structural lipids include one or a combination of cholesterol, cholestenol, spinasterol, coprostanol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid and α-tocopherol.

9. The lipid nanoparticle according to claim 1, characterized in that The nucleic acid includes one or a combination of siRNA, rRNA, DNA, aptamer, mRNA, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, and DNA enzyme.

10. The lipid nanoparticle according to claim 1, characterized in that The active ingredient includes one of mRNA-3927, mRNA-3705 or a combination thereof.

11. The lipid nanoparticle according to any one of claims 1 to 10, characterized in that The lipid nanoparticles include: ALC-0315, DMPC, cholesterol, PEG-DMG, and mRNA-3927.

12. Use of a pharmaceutical composition comprising the lipid nanoparticles according to any one of claims 1 to 11 in the preparation of a drug for preventing and / or treating rare inherited metabolic diseases of the liver.

13. The use according to claim 12, characterized in that: The rare inherited metabolic diseases of the liver include one or a combination of propionic acidemia, methylmalonic acidemia, tyrosinemia and ornithine transcarbamylase deficiency.

14. The lipid nanoparticle according to any one of claims 1 to 11 or the use according to claim 12, characterized in that: The lipid nanoparticles or drugs are administered by intramuscular injection.

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