Use of mannose-modified lipid nanoparticles

Abstract

The application belongs to the technical field of biology and particularly relates to application of mannose-modified lipid nanoparticles. The application discloses, for the first time, application of the mannose-modified lipid nanoparticles in preparation of a liver myeloid cell-targeted delivery system, and the mannose-modified lipid nanoparticles can be used as a liver myeloid cell-targeted delivery system to prepare a drug targeting liver myeloid cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of mannose-modified lipid nanoparticles. Background Technology

[0002] Chronic liver diseases, including viral hepatitis, liver fibrosis, and metabolic-associated fatty liver disease (MAFLD), can gradually worsen and even develop into cirrhosis and liver cancer if not properly treated and managed, seriously threatening human health and life. Currently, clinical treatment of liver diseases still faces significant challenges. Existing treatment methods mainly include etiological treatment, anti-inflammatory and hepatoprotective drugs, systemic anti-tumor therapy, and liver transplantation. Although etiological treatment can slow disease progression, it lacks a direct reversal effect on existing liver fibrosis and the disordered hepatic immune microenvironment. More importantly, the above therapies are all unable to target specific immune cell populations in the liver (such as Kupffer cells, Ly6C cells, etc.). hi While precise regulation of monocytes (monocytes) is possible, it cannot fundamentally prevent the progression of chronic inflammation towards fibrosis and tumor development. As the body's largest immune-protected organ, the liver's disease progression is highly dependent on myeloid immune cells (Kupffer cells, Ly6C cells, etc.). hi Imbalance in the function of monocytes and neutrophils is a key factor. These cells play a central regulatory role in chronic inflammation, fibrosis progression, and the formation of an immunosuppressive microenvironment. Their continuous release of inflammatory cytokines such as TGF-β, IL-1β, TNF-α, and type I interferon drives hepatic stellate cell activation and collagen deposition, contributing to the construction of an immunosuppressive microenvironment and promoting liver fibrosis and tumor progression. During the liver repair phase, hepatic myeloid immune cells can transform into anti-inflammatory and pro-repair phenotypes, promoting collagen degradation. Therefore, highly efficient and precise targeted regulation of hepatic myeloid immune cell function has become a crucial breakthrough for blocking or even reversing the progression of chronic liver disease.

[0003] Lipid nanoparticles (LNPs) are an ideal delivery platform for regulating hepatic myeloid cells: First, the unique physiological structure of the liver endows LNPs with a natural "first-pass" advantage, which, combined with the capture capability of the mononuclear phagocytic system (MPS), allows for passive enrichment of the payload in the hepatic myeloid region. Second, LNPs possess efficient intracellular escape capabilities, enabling precise delivery of nucleic acid drugs or immunomodulators to the cytoplasm of myeloid cells, reshaping cellular function at the transcriptional or translational level. However, LNPs still face two major bottlenecks in targeting myeloid cells: On the one hand, traditional LNPs are mainly non-specifically taken up by hepatocytes, making it difficult to effectively target hepatic myeloid cells; on the other hand, LNP components (especially ionizable lipids) easily activate Toll-like receptors and the NLRP3 inflammasome, triggering the release of pro-inflammatory factors such as IL-1β and TNF-α, which may exacerbate liver damage. Therefore, it is necessary to develop a delivery strategy that can target hepatic myeloid cells and / or inhibit the inflammatory response of LNPs themselves. Summary of the Invention

[0004] The first aspect of this invention is to provide the application of mannose-modified lipid nanoparticles.

[0005] The second aspect of the present invention is to provide the application of anti-inflammatory active substances.

[0006] A third aspect of the present invention is to provide a combination of active ingredients.

[0007] The fourth aspect of this invention aims to provide the application of the combination of active ingredients of the third aspect of this invention.

[0008] The fifth aspect of this invention is to provide a medicine.

[0009] The sixth aspect of this invention aims to provide a method.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the invention provides the use of mannose-modified lipid nanoparticles in any one of a1)-a2): a1) Preparation of a liver myeloid cell targeted delivery system; a2) Prepare drugs that target hepatic myeloid cells.

[0011] In some embodiments, mannose-modified lipid nanoparticles are used as a liver myeloid cell-targeted delivery system in the preparation of drugs targeting liver myeloid cells.

[0012] In some embodiments, the mannose is modified on the surface of the lipid nanoparticles; preferably, the mannose is attached to the surface of the lipid nanoparticles via a triazole bond.

[0013] In some embodiments, the raw materials for preparing the mannose-modified lipid nanoparticles include: neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids, and N-(2-azidoethyl)mannosamide.

[0014] In some embodiments, the neutral lipid may include, but is not limited to, one or more of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (distearylphosphatidylcholine, DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 2-dioleoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); more preferably, DSPC.

[0015] In some embodiments, the amphiphilic lipid may include, but is not limited to, one or more combinations of PEG-DMG, PEG-c-DMG, PEG-C14, PEG-c-DMA, PEG-DSPE, PEG-PE, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, PEG-DPG, PEG-s-DMG, DAA, PEG-c-DOMG, and GalNAc-PEG-DSG; more preferably, DMG-PEG2000.

[0016] In some embodiments, the cationic lipids may include, but are not limited to: (4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), octadecano-9-yl 8-((2-hydroxyethyl)(6-oxO-6-(undecyloxy)hexyl)amino)octanoate (SM-102), 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP), 313-[N-(N,N-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyloctadecylammonium (DDA), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), dipalmitoyl(C16:0)trimethylammonium propane (… One or more of the following: DPTAP, distearyltrimethylammonium propane (DSTAP), N-[1-(2,3-diallyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-propanetrioxy-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 4-(dimethylamino)butyrate heptadec-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); more preferably: SM-102.

[0017] In some embodiments, the steroidal lipids may include, but are not limited to, one or more of the following: alfalfa sterol, β-sitosterol, brassosterol, ergocalciferol, campesterol, cholesterol, coccidosterol, dehydrocholesterol, sterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, stigmasterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, physcosterol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid; more preferably, cholesterol.

[0018] In some embodiments, the method for preparing the mannose-modified lipid nanoparticles includes the following steps: b1) Neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids are mixed with an organic solvent (e.g., ethanol) to obtain an oil phase; b2) The oil phase is mixed with an aqueous solvent (e.g., an acidic aqueous solvent, preferably a sodium citrate / sodium acetic acid solution with a pH of 4-5) to obtain lipid nanoparticles; b3) The lipid nanoparticles are mixed with N-(2-azidoethyl)mannosamide and reacted to obtain the mannose-modified lipid nanoparticles.

[0019] In some embodiments, the molar ratio of neutral lipids, amphiphilic lipids, cationic lipids, and steroidal lipids in step b1) is (5-15):1.5:(40-60):(31-46); more preferably, it is about 10:1.5:50:38.5.

[0020] In some embodiments, the lipid nanoparticles and N-(2-azidoethyl)mannosamide in step b3) are in a molar ratio of 1:(1-2) based on the DBCO group of the lipid nanoparticles to N-(2-azidoethyl)mannosamide; more preferably, it is about 1:1.5.

[0021] In some embodiments, the liver myeloid cells include liver myeloid immune cells; more preferably, they include liver macrophages (Kupffer cells) and liver monocytes (Ly6C cells). hi At least one of the following: monocytes, liver neutrophils; further, liver macrophages or liver monocytes; and even further, liver macrophages.

[0022] In some embodiments, the drug targeting liver myeloid cells is used to prevent or treat liver myeloid cell-related diseases.

[0023] In some embodiments, the liver myeloid cell-related diseases include at least one of liver fibrosis, metabolic-associated steatohepatitis (MASH), metabolic-associated fatty liver disease (MAFLD), and liver cancer.

[0024] In some embodiments, the drug targeting liver myeloid cells comprises the mannose-modified lipid nanoparticles and a preventive or therapeutic active ingredient; wherein the mannose-modified lipid nanoparticles serve as a delivery system for the preventive or therapeutic active ingredient.

[0025] In some embodiments, the preventive or therapeutic active ingredient is an active ingredient for the prevention or treatment of diseases related to hepatic myeloid cells.

[0026] In some embodiments, the preventive or therapeutic active ingredient is a nucleic acid.

[0027] In some embodiments, the nucleic acid includes at least one of DNA and RNA; more preferably, it includes at least one of ssDNA, dsDNA, mRNA, lncRNA, siRNA, saRNA, shRNA, ASO, plasmid, circRNA, circDNA, miRNA, CRISPR-Cas, SamRNA, and antagomir; even more preferably, it includes at least one of mRNA, siRNA, miRNA, and CRISPR-Cas.

[0028] In some embodiments, the raw materials for preparing the drug targeting hepatic myeloid cells include: neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids, N-(2-azidoethyl)mannosamide, and preventive or therapeutic active ingredients.

[0029] In some embodiments, the method for preparing the drug targeting hepatic myeloid cells includes the following steps: c1) Neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids are mixed with an organic solvent (e.g., ethanol) to obtain an oil phase; c2) The preventive or therapeutic active ingredient is mixed with an aqueous solvent (e.g., an acidic aqueous solvent, preferably a sodium citrate / sodium acetic acid solution with a pH of 4-5) to obtain an aqueous phase; c3) The oil phase is mixed with the aqueous phase to obtain lipid nanoparticles containing preventive or therapeutic active ingredients; c4) The lipid nanoparticles containing preventive or therapeutic active ingredients are mixed with N-(2-azidoethyl)mannosamide and reacted to obtain the drug targeting liver myeloid cells.

[0030] In some embodiments, the molar ratio of neutral lipids, amphiphilic lipids, cationic lipids, and steroidal lipids in step c1) is (5-15):1.5:(40-60):(31-46); more preferably, it is about 10:1.5:50:38.5.

[0031] In some embodiments, the lipid nanoparticles containing preventive or therapeutic active ingredients described in step c4) and N-(2-azidoethyl)mannosamide are in a molar ratio of 1:(1-2) based on the DBCO group of the lipid nanoparticles containing preventive or therapeutic active ingredients to N-(2-azidoethyl)mannosamide; more preferably, it is about 1:1.5.

[0032] In this invention, the drug also includes pharmaceutically acceptable excipients.

[0033] In this invention, the dosage forms of the drugs are each independently either dosage forms suitable for children or dosage forms suitable for adults.

[0034] In this invention, the dosage form is selected from gastrointestinal dosage forms or non-gastrointestinal dosage forms.

[0035] In this invention, the gastrointestinal dosage form includes at least one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0036] In this invention, the non-gastrointestinal drug delivery dosage form includes at least one of the following: injectable dosage forms (e.g., injections, including various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections); respiratory dosage forms (e.g., sprays, aerosols, powder inhalers, etc.); skin dosage forms (e.g., topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal dosage forms (e.g., eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive tablets, films, etc.); and cavity dosage forms (e.g., suppositories, aerosols, effervescent tablets, drops, pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.); and further, injectable dosage forms.

[0037] A second aspect of the invention provides the use of the anti-inflammatory active substance in any one of d1)-d3): d1) Preparation of drugs to inhibit inflammation induced by lipid nanoparticles; d2) Prepare drugs for the prevention or treatment of lipid nanoparticle-related acute liver injury; d3) Prepare a medicament for the prevention or treatment of diseases related to a preventive or therapeutic active ingredient, wherein the anti-inflammatory active substance is used in combination with lipid nanoparticles containing the preventive or therapeutic active ingredient.

[0038] In this invention, the anti-inflammatory active substance includes: dexamethasone or a pharmaceutically acceptable salt, solvate or ester thereof.

[0039] In some embodiments, the lipid nanoparticles described in d1)-d3) are mannose-modified lipid nanoparticles as described in the first aspect of the present invention.

[0040] In some embodiments, the disease associated with the preventive or therapeutic active ingredient described in d3) is a liver myeloid cell-related disease as described in the first aspect of the invention.

[0041] In some embodiments, the lipid nanoparticles containing the preventive or therapeutic active ingredient described in d3) are drugs targeting hepatic myeloid cells in the first aspect of the present invention.

[0042] In some embodiments, the anti-inflammatory active substance described in d3) is not contained within the lipid nanoparticles.

[0043] In this invention, the pharmaceutically acceptable salt comprises at least one of a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0044] In this invention, the metal salt comprises at least one of alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (e.g., calcium salts, magnesium salts, barium salts, etc.), and aluminum salts.

[0045] In this invention, the salt formed with an organic base includes salts formed with one or more of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0046] In this invention, the salt formed with the inorganic acid includes salts formed with one or more of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0047] In this invention, the salt formed with organic acids includes salts formed with one or more of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0048] In this invention, the salt formed with the basic amino acid includes salts formed with one or more of the following basic amino acids: arginine, lysine, ornithine.

[0049] In this invention, the salt formed with acidic amino acids includes salts formed with one or more of the following acidic amino acids: aspartic acid and glutamic acid.

[0050] A third aspect of the present invention provides an active ingredient combination comprising a first active ingredient and a second active ingredient: The first active ingredient includes: anti-inflammatory active substances; The second active ingredient includes: lipid nanoparticles containing preventive or therapeutic active ingredients.

[0051] In some embodiments, the lipid nanoparticles are mannose-modified lipid nanoparticles as described in the first aspect of the present invention.

[0052] In some embodiments, the lipid nanoparticles containing preventive or therapeutic active ingredients are drugs targeting hepatic myeloid cells as described in the first aspect of the present invention.

[0053] In some embodiments, the first active ingredient is not located within the lipid nanoparticles of the second active ingredient.

[0054] In some embodiments, the mass ratio of the first active ingredient to the second active ingredient is (5-15):1; more preferably (8-12):1; and even more preferably (9-11):1.

[0055] In some embodiments, the first active ingredient and the second active ingredient exist independently or in combination.

[0056] In some embodiments, the first active ingredient and the second active ingredient are applied simultaneously, separately, or sequentially.

[0057] A fourth aspect of the invention provides the use of the combination of active ingredients of the third aspect of the invention in the preparation of a medicament for preventing or treating diseases related to the preventive or therapeutic active ingredient in the second active ingredient.

[0058] In some embodiments, the disease associated with the preventive or therapeutic active ingredient in the second active ingredient is a liver myeloid cell-related disease as described in the first aspect of the invention.

[0059] A fifth aspect of the present invention provides a medicament comprising a combination of active ingredients from the third aspect of the present invention.

[0060] In some embodiments, the drug is used to prevent or treat diseases associated with the preventive or therapeutic active ingredient in the second active ingredient.

[0061] In some embodiments, the disease associated with the preventive or therapeutic active ingredient in the second active ingredient is a liver myeloid cell-related disease as described in the first aspect of the invention.

[0062] A sixth aspect of the present invention provides a method for inhibiting inflammation caused by lipid nanoparticles, or for preventing or treating lipid nanoparticle-related acute liver injury, by administering an effective amount of an anti-inflammatory active substance to a subject.

[0063] A method for preventing or treating a disease, comprising administering to a subject an effective amount of a combination of active ingredients of the third aspect of the present invention or a drug of the fifth aspect; wherein the disease is a disease related to the preventive or therapeutic active ingredient in the second active ingredient.

[0064] In some implementations, the subject may include mammals, such as humans or non-human mammals.

[0065] In some embodiments, the non-human mammals may include, but are not limited to, non-human primates (e.g., monkeys, orangutans), mice, rats, hamsters, gerbils, cats, dogs, guinea pigs, rabbits, horses, sheep, cattle, pigs, etc.

[0066] The beneficial effects of this invention are: This invention discloses for the first time the application of mannose-modified lipid nanoparticles in the preparation of a liver myeloid cell targeted delivery system, which can be used as a liver myeloid cell targeted delivery system to prepare drugs targeting liver myeloid cells.

[0067] This invention discloses for the first time the application of anti-inflammatory active substances in inhibiting inflammation caused by lipid nanoparticles or in preventing or treating lipid nanoparticle-related acute liver injury. Based on this, it can be used in combination with lipid nanoparticles containing preventive or therapeutic active ingredients to prevent or treat diseases related to the preventive or therapeutic active ingredients.

[0068] This invention discloses for the first time an active ingredient combination comprising a first active ingredient and a second active ingredient: the first active ingredient comprises an anti-inflammatory active substance; the second active ingredient comprises lipid nanoparticles containing preventive or therapeutic active ingredients; which can inhibit the excessive inflammatory response caused by LNP itself while targeting target cells; Furthermore, by limiting the first active ingredient to not being within the lipid nanoparticles of the second active ingredient; This combination of active ingredients has the following advantages: 1) This is the first time that a synergistic scheme of "targeted LNP + dexamethasone combination" has been proposed, overcoming the technical defects of co-encapsulation. Existing technologies typically encapsulate anti-inflammatory active substances within LNPs, which can easily lead to lipid membrane instability, premature drug leakage, and increased formulation complexity. This invention explicitly uses the anti-inflammatory active substance as a drug delivery component independent of the LNP, without altering the LNP's formulation, particle size, or encapsulation efficiency. This avoids interference from hormone encapsulation on nucleic acid loading and LNP self-assembly, while preserving the high encapsulation efficiency (≥90%) and good colloidal stability inherent in mannose-modified LNPs.

[0069] Furthermore, by specifying the lipid nanoparticles as mannose-modified lipid nanoparticles, this combination of active ingredients offers the following advantages: 2) While achieving highly efficient targeting of hepatic myeloid cells, it significantly inhibits the inflammatory response induced by LNP itself. Mannose-modified lipid nanoparticles were used to target liver Kupffer cells and Ly6C. hi The monocytes take up the LNP efficiently, with a transfection efficiency of over 65%. At the same time, the free anti-inflammatory active substance selectively blocks the innate immune activation (IL-1β / TNF-α release) induced by the uptake of LNP by myeloid cells by inhibiting the NF-κB pathway, without affecting the uptake of LNP and intracellular nucleic acid release, forming a synergistic effect of "targeted enhancement-inflammation suppression". This effect cannot be achieved by administering LNP modified with mannose alone or by administering a single anti-inflammatory active substance. 3) It eliminates acute liver injury caused by LNP, demonstrating significant safety advantages. In normal mice, 24 hours after administration, mannose-modified LNP alone (G3 group) caused inflammatory cell infiltration in the portal area; while the proposed method (G4 group) showed intact liver lobule structure and significantly reduced inflammatory infiltration, and the liver tissue pathological score was not statistically different from the blank control group, proving that the proposed method can effectively eliminate the acute hepatotoxicity caused by the "self-adjuvant effect" of LNP, and provides a safety guarantee for its repeated clinical administration and long-term treatment of chronic liver disease. 4) Wide applicability and platform-based delivery value The mannose-modified LNP of this invention can load different nucleic acid drugs such as mRNA, siRNA, miRNA or CRISPR-Cas system, and combine with anti-inflammatory active substances to form a universal "targeted delivery + anti-inflammatory protection" solution, which is particularly suitable for the prevention and treatment of liver fibrosis, MASH, liver cancer and LNP-related acute liver injury, and has good prospects for clinical translation. Attached Figure Description

[0070] Figure 1 The particle size distribution of unmodified mannose-LNP-EGFP and mannose-modified mannose-LNP-EGFP in Example 2 is shown: A shows the particle size distribution of unmodified mannose-LNP-EGFP in Example 2; B shows the particle size distribution of mannose-modified mannose-LNP-EGFP.

[0071] Figure 2 Potentiograms of unmodified mannose-LNP-EGFP and mannose-modified mannose-LNP-EGFP in Example 2 are shown: A shows the potential diagram of unmodified mannose-LNP-EGFP in Example 2; B shows the potential diagram of mannose-modified mannose-LNP-EGFP in Example 2.

[0072] Figure 3 The transfection efficiency of mannose-modified-LNP-EGFP in MAC cells and the viability and yield of human Mac cells after transfection are shown in Example 3: A shows the viability of human Mac cells after transfection with mannose-modified-LNP-EGFP; B shows the yield of human Mac cells after transfection with mannose-modified-LNP-EGFP; C shows the transfection efficiency of mannose-modified-LNP-EGFP in MAC cells.

[0073] Figure 4The liver targeting of mannose-modified LNP-Luci in Example 4 is illustrated in: A shows the bioluminescence signal intensity of the major organs of mice injected with mannose-modified LNP-Luci; B shows the statistical results of the bioluminescence signal intensity of the major organs of mice injected with mannose-modified LNP-Luci; C shows the bioluminescence signal intensity of the liver and spleen of mice injected with mannose-modified LNP-Luci; and D shows the statistical results of the bioluminescence signal intensity of the liver and spleen of mice injected with mannose-modified LNP-Luci.

[0074] Figure 5 The distribution of mannose-modified-LNP-EGFP in mouse liver immune cells is shown in Example 5.

[0075] Figure 6 The expression of inflammatory cytokines in the serum of mice in groups G1, G2, G3, and G4 in Example 6 is shown.

[0076] Figure 7 The following shows the ALT / AST levels in the serum of mice in groups G1, G2, G3, and G4, and the H&E staining in the liver tissue of mice in groups G1, G2, G3, and G4 in Example 7: A shows the ALT / AST levels in the serum of mice in groups G1, G2, G3, and G4 in Example 7; B shows the H&E staining in the liver tissue of mice in groups G1, G2, G3, and G4.

[0077] Figure 8 The EGFP positivity rate of mouse liver immune cells in groups G1, G2, G3, and G4 in Example 7 is shown. Detailed Implementation

[0078] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in many publications.

[0079] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0080] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0081] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0082] Those skilled in the art will understand that when a numerical range is listed herein, it is intended to indicate that any intermediate value or range of values ​​within the listed numerical range is also within the scope of this invention.

[0083] As used herein, the terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations thereof, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0084] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0085] Example 1: Preparation of mannose-modified LNPs targeting hepatic myeloid cells This embodiment uses microfluidic mixing technology to prepare LNPs. The specific steps are as follows: 1. Dissolve the mRNA encoding EGFP protein (Shenji, 11011-CAP-1) in a pH 4.0 sodium citrate / sodium citrate solution to obtain an aqueous mRNA solution. 2. Lipid preparation: Prepare a lipid solution by dissolving it in ethanol solution according to a certain ratio (lipid molar ratio of SM-102:DSPC:cholesterol:DMG-PEG 2000 = 50:10:38.5:1.5) to obtain a lipid ethanol solution. 3. Draw 15 mM lipid ethanol solution and 0.05 μg / μL mRNA aqueous solution into the solution using a syringe, mix them using a chip, and mix the two phases at a volume ratio of 1 (ethanol phase): 3 (buffer phase) at 12 mL / min to obtain LNPs. 4. Dialyze overnight into 1xTBS, and then concentrate to the target volume by ultrafiltration. At this point, it is mannose unmodified-LNP-EGFP.

[0086] N-(2-azidoethyl)mannosamide (Man-N3, purchased from Xi'an Qiyue Biotechnology, Q-0028419) was dissolved in DMSO to prepare a 20 mM stock solution. The theoretical concentration of DBCO groups on the surface of the dialyzed LNP suspension (i.e., the above-mentioned unmodified mannose-LNP-EGFP) was calculated. A 1.5-fold molar excess of Man-N3 solution (relative to the DBCO groups) was added to the LNP suspension. The reaction was carried out with gentle stirring for 2 h at room temperature and in the dark. The reaction proceeded spontaneously via copper-free click chemistry (SPAAC), with mannose linked to the LNP surface via stable triazole bonds. The resulting solution was purified by centrifugation (4000 g, 10 min) using a 100 kDa molecular weight cutoff ultrafiltration centrifuge tube, repeated three times to completely remove unreacted Man-N3, DMSO, and any potential aggregates. Finally, the LNP was resuspended in PBS and filtered through a 0.22 μm sterile filter membrane to obtain the final product, mannose-modified LNP-EGFP, which was aliquoted and stored at -80℃ for later use.

[0087] Example 2: Physicochemical characterization of mannose-modified LNPs To confirm that mannose modification does not affect the basic properties of LNPs, the particle size and PDI of the LNPs (unmodified mannose-LNP-EGFP and modified mannose-LNP-EGFP) prepared in Example 1 were measured using a particle size analyzer (Malvin). The results showed that the hydrated particle sizes of unmodified mannose-LNP-EGFP and modified mannose-LNP-EGFP were 78.5 nm and 73.49 nm, respectively, and the polydispersity index (PDI) were 0.08 and 0.03, respectively. Figure 1 The zeta potentials measured in PBS were -1.52 mV and -1.95 mV, both near-neutral, which is beneficial for long-term circulation in vivo. Figure 2 Using Quant-iT TM The RiboGreen quantitative reagent kit was used to detect the encapsulation efficiency of the LNPs (mannose-unmodified-LNP-EGFP and mannose-modified-LNP-EGFP) prepared in Example 1. Small amounts of the LNP formulation were added to Triton X-100 for lysis (total nucleic acid measurement) and for non-lysis (free nucleic acid measurement), respectively, and then bound to the dye. The encapsulation efficiency was calculated based on the fluorescence value. The encapsulation efficiencies of mannose-unmodified-LNP-EGFP and mannose-modified-LNP-EGFP were 94.66% and 96.87%, respectively, indicating that the preparation process was highly efficient.

[0088] Example 3: In vitro safety evaluation and in vitro PBMC targeting validation of mannose-modified LNPs To verify the in vitro safety of the mannose-modified LNPs of this invention, human macrophages (MACs) were cultured at a density of 1.5 × 10⁶ cells per well.7 -3×10 7 Cells were seeded at a density of 1 × 10⁶ cells / year in G-REX24 cells and cultured at 37°C and 5% CO₂ for 24 h. LNPs (unmodified mannose-LNP-EGFP and mannose-modified LNP-EGFP) prepared in Example 1 were used. Group G1 consisted of cells without LNP treatment (UTD), and Group G2 consisted of cells seeded at a density of 1 × 10⁶ cells / year. 6 MAC cells were treated with 1 μg of unmodified mannose-LNP-EGFP, with each G3 group receiving 1 × 10⁻⁶ cells. 6 MAC cells were transfected with 1 μg of mannose-modified LNP-EGFP for 24 h, and the cell viability and yield of each group were collected and statistically analyzed. Figure 3 Figures A and 3B show that, compared with UTD, there was no statistically significant difference in the viability and yield of human Mac cells in groups G2 and G3. The positivity rate of EGFP in these MAC cells was detected by flow cytometry (FACS). Figure 3 As shown in C, the positive rate of EGFP in MAC cells transfected with mannose-modified-LNP-EGFP was over 95% (96.3%), which was significantly higher than that in MAC cells transfected with unmodified mannose-LNP-EGFP (51.6%).

[0089] Example 4: In vivo distribution assessment of mannose-modified LNPs To verify the in vivo distribution of the mannose-modified LNPs described in this invention, luciferase messenger RNA (Novizan, DD4511-01) was selected as the model nucleic acid (Luci-mRNA) for in vivo distribution studies. This embodiment included two groups: G1, unmodified mannose-LNP-Luci group; and G2, modified mannose-LNP-Luci group.

[0090] Unmodified mannose-LNP-Luci and mannose-modified mannose-LNP-Luci were administered to 8-week-old C57BL / 6J mice via tail vein injection at a dose of 0.5 mg / kg. The preparation methods for unmodified mannose-LNP-Luci and mannose-modified mannose-LNP-Luci were the same as in Example 1, except that the mRNA encoding EGFP protein was replaced with Luci-mRNA. Six hours after injection, the mice were anesthetized with 2% isoflurane, and then D-fluorescein potassium salt (purchased from PerkinElmer) was subcutaneously injected at a dose of 150 mg / kg. The major organs were imaged using the IVIS Lumina III in vivo imaging system (PerkinElmer), and the bioluminescence signal intensity was semi-quantitatively analyzed based on the acquired images.

[0091] The results are as follows Figure 4As shown: Compared with group G1, after injection of mannose-modified-LNP-Luci, the bioluminescent signal in group G2 was mainly enriched in the liver of mice, and the difference was significant (P<0.01). The mannose-modified-LNP of the present invention can significantly enhance its targeting effect in the liver.

[0092] Example 5: Evaluation of in vivo delivery efficiency and liver immune cell targeting specificity of mannose-modified LNPs To verify the targeting effect of the mannose-modified LNP on myeloid liver cells in vivo, flow cytometry was used to study the targeting specificity and delivery efficiency of different liver cell types in vivo.

[0093] Wild-type C57BL / 6J mice aged 6-8 weeks were randomly divided into 3 groups (n=3): G1, mannose-modified LNP-empty group; G2, mannose-unmodified LNP-EGFP group; and G3, mannose-modified LNP-EGFP group. LNP (mannose-modified LNP-empty, mannose-unmodified LNP-EGFP, and mannose-modified LNP-EGFP) were injected via tail vein, at a rate of 0.5 mg / kg. The mannose-unmodified LNP-EGFP and mannose-modified LNP-EGFP were the same as those in Example 1. The preparation method of mannose-modified LNP-empty was the same as that of mannose-modified LNP-EGFP, except that it did not contain mRNA encoding the EGFP protein. Six hours after administration, the mice were euthanized and perfused with the heart at a flow rate of 5-7 mL / min for about 3 minutes until the liver turned white and most of the blood cells were washed away.

[0094] Preparation of liver single-cell suspension: The liver was removed and minced to 2-4 mm using scissors. The tissue was transferred to a 50 mL centrifuge tube, and collagenase IV (purchased from Sigma) was added to a final concentration of 1 mg / mL. The tissue was placed in a shaker and digested at 120 rpm and 37°C for 40 min. Then, 1640 medium containing 2% fetal bovine serum (FBS, purchased from Gibco) was added to terminate the digestion. The mixture was filtered through a 70 μm cell sieve to obtain a single-cell suspension. The cell suspension was centrifuged at 50 × g and 4°C for 3 min, and the supernatant (mainly containing non-parenchymal cells, NPCs) was carefully aspirated. The supernatant (NPC fraction) was centrifuged again at 400 × g and 4°C for 5 min, and the supernatant was discarded to obtain the NPC pellet. The pellet was treated with erythrocyte lysis buffer (purchased from Biolegend) for 10 min to lyse the erythrocytes, resuspended in PBS containing 2% FBS, and counted for later use.

[0095] Flow cytometry antibody staining: Add 2 μL of purified anti-mouse CD16 / 32 antibody (purchased from Biolegend) to each sample, incubate at room temperature in the dark for 10 min, then add anti-Mouse-CD45 antibody, anti-Mouse-F4 / 80 antibody, anti-Mouse-CD11b antibody, anti-Mouse-Ly-6C antibody, anti-Mouse-Ly-6G antibody (the above antibodies were purchased from BD), anti-Mouse-CD3 antibody, anti-Mouse-CD19 antibody, and anti-Mouse-NK1.1 antibody (the above antibodies were purchased from Biolegend), incubate at 4°C in the dark for 30 min, centrifuge at 400 g for 5 min, wash once with PBS, and then perform flow cytometry detection.

[0096] Flow cytometry results showed that, compared with the G2 unmodified mannose-LNP-EGFP group, the proportion of EGFP+ in mouse liver myeloid cells in the G3 group was significantly increased, especially in mouse liver macrophages where the proportion of positive cells exceeded 60%. Furthermore, the proportion of EGFP+ in T, B, and NK lymphocytes was significantly lower than that in the G2 group. The delivery efficiency of mannose-modified LNP-EGFP in CD45- cells (hepatocytes) was extremely low, approximately 5%. This indicates that the mannose-modified LNP described in this invention can specifically and efficiently deliver nucleic acids to liver myeloid cells. Figure 5 ).

[0097] Example 6: Evaluation of the anti-inflammatory effect of mannose-modified LNP+DXMS To verify the inhibitory effect of the combination of mannose-modified LNP and dexamethasone (mannose-modified LNP + DXMS) on systemic inflammatory response, this embodiment used flow cytometry (CBA) to detect the levels of inflammatory factors in the peripheral blood of mice. The specific steps are as follows: Wild-type male C57BL / 6J mice aged 6-8 weeks were randomly divided into 4 groups (n=5): G1, PBS group; G2, DXMS; G3, mannose-modified LNP-EGFP group (mannose-modified LNP); G4, mannose-modified LNP-EGFP + dexamethasone group (mannose-modified LNP + DXMS). Groups G2 and G4 were intraperitoneally injected with 5 mg / kg DXMS one day prior to treatment. On the second day, groups G3 and G4 were injected with 0.5 mg / kg of the mannose-modified LNP-EGFP prepared in Example 1 via tail vein injection. Twenty-four hours after administration (mannose-modified LNP-EGFP), blood was collected from the posterior orbital venous plexus, and serum was separated. The concentrations of IL-6, MCP-1, and TNF-α in the serum were detected using the BD CBAMouse Inflammation Kit. Data were analyzed using FCAP Array Software. Results showed that group G3 induced an inflammatory response, with serum IL-6, MCP-1, and TNF-α concentrations surging to 39.72 pg / mL, 210.7 pg / mL, and 182.2 pg / mL, respectively. In contrast, the IL-6 and TNF-α levels in group G4 were only 21.9 pg / mL, 71.8 pg / mL, and 62.8 pg / mL, respectively. The results demonstrate that the mannose-modified LNP+DXMS successfully overcomes the "self-adjuvant effect" of traditional LNPs and single-target LNPs, achieving a "delivery-as-anti-inflammatory" therapeutic effect, and greatly improving the safety of LNP delivery systems in repeated administration in chronic liver disease. Figure 6 ).

[0098] Example 7: Safety assessment of the mannose-modified LNP+DXMS combination For the mice in Example 6 above, the experimental endpoint was achieved by collecting mouse serum for biochemical index detection; mouse liver tissue was taken, fixed, and subjected to H&E staining.

[0099] from Figure 7As shown in Figure A, 24 h after drug administration, the serum ALT and AST levels in mice in group G4 (mannose-modified LNP + DXMS) were significantly lower than those in group G3 (P < 0.01), and remained within the normal range. H&E staining results showed that hepatocyte cords in groups G1 and G2 were neatly arranged, with no obvious inflammatory cell infiltration in the portal areas and around the central vein; in group G3 (mannose-modified LNP), inflammatory cell aggregation, mainly neutrophils and monocytes, was observed in the portal areas and sinusoids, significantly higher than in group G1. In stark contrast, the liver tissue structure in group G4 was basically restored to normal, with significantly reduced inflammatory cell infiltration, mild hepatocyte degeneration, and no statistically significant difference in pathological score compared to group G1. Figure 7 B).

[0100] Flow cytometry results showed that, compared with the G1 and G2 groups, the proportion of EGFP+ in the myeloid cells of the liver of mice in the G3 and G4 groups was significantly increased, and the proportion of EGFP-positive cells in liver macrophages was higher than 60%. Figure 8 The above results indicate that the mannose-modified LNP+DXMS drug combination can effectively block the acute damage to normal liver tissue caused by LNP during the peak period of inflammation (24 h), thereby achieving the safe characteristic of "targeted delivery without harming normal tissue".

[0101] The technical solutions disclosed herein are not limited to the specific embodiments described above. Any technical modifications made based on the technical solutions disclosed herein shall fall within the protection scope of this disclosure.

Claims

1. Application of mannose-modified lipid nanoparticles in any of a1)-a2): a1) Preparation of a liver myeloid cell targeted delivery system; a2) Prepare drugs that target hepatic myeloid cells.

2. The application according to claim 1, characterized in that, The liver myeloid cells include liver myeloid immune cells; more preferably, they include at least one of liver macrophages, liver monocytes, and liver neutrophils; even more preferably, they are liver macrophages or liver monocytes. Further, liver macrophages; Preferably, the raw materials for preparing the mannose-modified lipid nanoparticles include: neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids, and N-(2-azidoethyl)mannosamide; Preferably, the drug targeting hepatic myeloid cells is used to prevent or treat hepatic myeloid cell-related diseases; Preferably, the liver myeloid cell-related diseases include at least one of liver fibrosis, metabolic-associated steatohepatitis (MASH), metabolic-associated fatty liver disease (MAFLD), and liver cancer; Preferably, the drug targeting liver myeloid cells comprises the mannose-modified lipid nanoparticles and a preventive or therapeutic active ingredient; Preferably, the preventive or therapeutic active ingredient is a nucleic acid; Preferably, the raw materials for preparing the drug targeting hepatic myeloid cells include: neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids, N-(2-azidoethyl)mannosamide, and preventive or therapeutic active ingredients.

3. Application of anti-inflammatory active substances in any of the following stages (d1)-d3): d1) Preparation of drugs to inhibit inflammation induced by lipid nanoparticles; d2) Prepare drugs for the prevention or treatment of lipid nanoparticle-related acute liver injury; d3) Prepare a medicament for the prevention or treatment of diseases related to a preventive or therapeutic active ingredient, wherein the anti-inflammatory active substance is used in combination with lipid nanoparticles containing the preventive or therapeutic active ingredient.

4. The application according to claim 3, characterized in that, The anti-inflammatory active substances include: dexamethasone or its pharmaceutically acceptable salts, solvates or esters; Preferably, the lipid nanoparticles in d1)-d3) are mannose-modified lipid nanoparticles according to any one of claims 1-2; Preferably, the disease associated with the preventive or therapeutic active ingredient in d3) is the liver myeloid cell-related disease as described in claim 2; Preferably, the lipid nanoparticles containing the preventive or therapeutic active ingredient described in d3) are the drugs targeting hepatic myeloid cells as described in any one of claims 1-2; Preferably, the anti-inflammatory active substance described in d3) is not contained within the lipid nanoparticles.

5. A combination of active ingredients, comprising a first active ingredient and a second active ingredient: The first active ingredient includes: Anti-inflammatory active substances; The second active ingredient includes: lipid nanoparticles containing preventive or therapeutic active ingredients.

6. The active ingredient combination according to claim 5, characterized in that, The anti-inflammatory active substances include: dexamethasone or its pharmaceutically acceptable salts, solvates or esters; Preferably, the lipid nanoparticles are mannose-modified lipid nanoparticles according to any one of claims 1-2; Preferably, the lipid nanoparticles containing the preventive or therapeutic active ingredient are drugs targeting hepatic myeloid cells as described in any one of claims 1-2; Preferably, the first active ingredient is not located within the lipid nanoparticles of the second active ingredient.

7. The combination of active ingredients according to any one of claims 5-6, characterized in that, The mass ratio of the first active ingredient to the second active ingredient is (5-15):1; more preferably (8-12):

1. Preferably, the first active ingredient and the second active ingredient exist independently or in combination; Preferably, the first active ingredient and the second active ingredient are applied simultaneously, separately, or sequentially.

8. The use of the combination of active ingredients according to any one of claims 5-7 in the preparation of a medicament for the prevention or treatment of diseases related to the preventive or therapeutic active ingredient in the second active ingredient; Preferably, the disease associated with the preventive or therapeutic active ingredient in the second active ingredient is a liver myeloid cell-related disease as described in claim 2.

9. A medicament comprising the combination of active ingredients as described in any one of claims 5-7.

10. The application or drug according to any one of claims 1-4 and 8-9, characterized in that, The drug also includes pharmaceutically acceptable excipients; Preferably, the dosage forms of the drugs are each independently a dosage form suitable for children or a dosage form suitable for adults; Preferably, the dosage form is selected from gastrointestinal dosage forms or non-gastrointestinal dosage forms.

Images