An imidazole-ionizable lipid-based lipid nanoparticle, its preparation method and application

Abstract

The present invention provides an imidazole-ionizable lipid-based lipid nanoparticle and its preparation method and application. The blank lipid nanoparticles prepared by the present invention have strong designability, biodegradability and high in vitro and in vivo delivery efficiency. As a drug carrier for siRNA delivery, it is superior to currently marketed products at the in vitro level and shows good therapeutic effects in in vivo tumor treatment, and can be used as a new method for delivering nucleic acids or anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to an imidazole-ionizable lipid-based lipid nanoparticle and its preparation method and application, belonging to the technical field of drug delivery. Background Art

[0002] Nucleic acid drugs, including mRNA, DNA, miRNA, and siRNA, etc., are a class of emerging drugs. In principle, nucleic acid drugs can target all genes. They are applicable to targeting undruggable proteins. Therefore, they show the potential to treat various diseases. However, nucleic acid drugs are polyvalent anions and highly hydrophilic molecules. They are hardly taken up by cells and are easily degraded by nucleases in the blood. Therefore, they require delivery carriers to enter cells and play their roles.

[0003] Lipid nanoparticles (LNPs) have become the most promising carriers for clinical applications among various non-viral carriers due to their excellent biocompatibility, biodegradability, low toxicity, low immunogenicity, structural flexibility, and ease of large-scale preparation. In the past few decades, a variety of LNPs have been reported for intracellular delivery of nucleic acid drugs, and many LNP drug formulations have been approved for clinical use. LNPs have the ability to control the location and time of drug delivery in vivo, so they can be used for the treatment of various diseases. The breakthroughs of LNP-based mRNA vaccines and other therapies have demonstrated the potential of the LNP system, especially the ionizable (cationic) lipid LNP system, for delivering therapeutic nucleic acids. Therefore, it has triggered a new wave of development, that is, the ionizable lipid-based LNP system for delivering various therapeutic nucleic acids to prevent and treat human diseases that cannot be treated by conventional treatment regimens.

[0004] Many ionizable lipids with ionizable amino headgroups have been developed as key components of LNP formulations. These ionizable lipids usually contain an amino head and have an acid dissociation constant (pKa) less than 7; thus, they are protonated and positively charged at acidic pH (pH < 6.0) and neutral under physiological conditions (pH = 7.4). They can form LNPs with a total surface charge close to neutral, and compared with cationic delivery systems after systemic administration, they exhibit reduced toxicity and extended circulation time, allowing them to enter more tissues. In acidified endosomes, the ionizable lipids are protonated, and the resulting positively charged lipids can interact with the negatively charged endosomal membrane, leading to endosomal membrane instability and release of the RNA cargo into the cytoplasm. One or more domains of the headgroup, linker, or hydrophobic tail of the ionizable lipid can be optimized by combining iterative screening and combinatorial chemistry. Ionizable lipids with a specific pKa can provide high-efficiency encapsulation and endosomal escape of nucleic acid drugs, and highly efficient ionizable lipids provide better immune responses for RNA-LNP vaccines while maintaining low toxicity. Therefore, the research objective of this project aims to design and synthesize novel ionizable cationic lipids through the relationship between pKa and structure, and efficiently encapsulate and deliver mRNA for the treatment of diseases.

[0005] Since nucleic acid drugs can specifically inhibit the function of target proteins, they have received great attention as anticancer agents. Despite many efforts to improve the delivery of nucleic acid drugs for cancer treatment, no anticancer nucleic acid drugs have been launched. Perhaps due to delivery problems, the anticancer effects of RNA-based therapies may still be insufficient. Although LNP-mediated delivery of RNA to liver tumors is promising because LNPs coated with ApoE in the blood are expected to be internalized into liver cancer cells through the ApoE-LDLR pathway, delivery systems for RNA to other cancer tissues are still being explored. Designing and synthesizing ionizable lipids with low toxicity, high efficiency, and targeting properties will open the way for constructing innovative LNP systems for cancer treatment. Summary of the Invention

[0006] To address the above technical problems, the present invention provides a compound represented by formula (I):

[0007]

[0008] Wherein, M 1 、M 2 are the same or different and are independently selected from -CH 2 -, -(CH 2 ) m -, -CH(OH)-, -CH 2 O-, -OC(=O)-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, -C(=O)S-; m is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0009] G 1 、G 2 are the same as or different from each other and are independently selected from -CH 2 -, -CH(OH)-, -CH 2 O-, -OC(=O)-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, -C(=O)S-;

[0010] E 1 、E 2 are the same as or different from each other and are independently selected from -CH 2 -, -CH(OH)-, -CH 2 O-, -OC(=O)-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, -C(=O)S-;

[0011] L 1 is wherein A 1 、A 2 、A 3 does not exist, or is the same as or different from each other and is independently selected from, unsubstituted or optionally substituted by one or more C 1-10 alkyl-substituted following groups: sub-C 1-6 alkyl, sub-C 1-6 alkyloxy, sub-C 2-6 alkenyl, sub-C 3-8 cycloalkyl, sub-3-8 membered heterocyclic group, sub-C 6-10 aryl, sub-5-10 membered heteroaryl, NH; n1 and n2 are the same as or different from each other and are independently selected from 0, 1, 2, 3, 4, 5 or 6;

[0012] According to an embodiment of the present invention, L 1 is selected from L-1, L-2, L-3, L-4, L-5, L-6, L-7, L-8, L-9, L-10, L-11 or L-12;

[0013]

[0014] R 1 is selected from -H or -NH 2 ;

[0015] J 1 is selected from -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2O-, -CH(OH)-, -CH(NH 2 )-;

[0016] n is a number selected from 2 - 22, such as 3, 5, 7, 10, 12, and 13, preferably 3, 7, or 10.

[0017] According to an embodiment of the present invention, the compound shown in formula (I) is selected from compounds having the following structures:

[0018]

[0019] The present invention provides a blank lipid nanoparticle, comprising a compound shown in formula (I), phospholipid, cholesterol (CHOL), and PEG lipid;

[0020] According to an embodiment of the present invention, the phospholipid is selected from at least one of DSPC, DPPC, POPC, DOPE, and DEPC, preferably distearoyl phosphatidylcholine (DSPC);

[0021] According to an embodiment of the present invention, the PEG lipid is selected from at least one of DSPE - PEG 2000, MG - PEG 2000, DPPE - PEG 2000, and DMA - PEG 2000, preferably 1,2 - dimyristoyl - rac - glycerol - 3 - methoxypolyethylene glycol 2000 (DMG - PEG 2000).

[0022] According to an embodiment of the present invention, the molar fraction of the compound shown in formula (I) is 20 - 60%, such as 40 - 55%, such as 45%, 48%, 50%, 52%;

[0023] According to an embodiment of the present invention, the molar fraction of the PEG lipid is 0.1 - 5%, such as 0.5 - 3%, such as 0.8%, 1%, 1.2%, 1.5%, 2%, 2.5%;

[0024] According to an embodiment of the present invention, the molar fraction of the phospholipid is 5 - 20%, such as 8 - 15%, such as 9%, 10%, 11%, 12%;

[0025] According to an embodiment of the present invention, the molar fraction of the cholesterol is 15 - 55%, such as 30 - 50%, such as 32%, 35%, 36%, 38%, 40%, 42%, 45%.

[0026] According to an embodiment of the present invention, the molar ratio of the compound shown in formula (I), phospholipid, cholesterol, and PEG lipid is 50:10:38.5:1.5.

[0027] The present invention also provides a method for preparing the blank lipid nanoparticle, comprising the following steps:

[0028] (1) Dissolve the compound shown in formula (I), cholesterol, PEG lipid, and phospholipid in an organic solvent to obtain a lipid stock solution;

[0029] (2) Inject the lipid stock solution into a sodium citrate buffer solution to obtain the blank lipid nanoparticles;

[0030] According to an embodiment of the present invention, the organic solvent in step (1) is selected from alcohol solvents, for example, at least one selected from methanol, ethanol, and isopropanol;

[0031] According to an embodiment of the present invention, the volume ratio of the sodium citrate buffer solution to the lipid stock solution in step (2) is (1 - 6):1, for example, (2 - 5):1, such as 3:1 or 4:1.

[0032] The present invention also provides the use of the blank lipid nanoparticles as a drug carrier.

[0033] The present invention also provides a drug-loaded lipid nanoparticle composition, comprising the blank lipid nanoparticles and a drug.

[0034] According to an embodiment of the present invention, the mass ratio of the blank lipid nanoparticles to the drug is (5 - 30):1, for example, (10 - 20):1, preferably 10:1, 15:1, 20:1, 25:1, 30:1, 40:1.

[0035] According to an embodiment of the present invention, the drug can be a nucleic acid drug, and the nucleic acid molecule can be selected from at least one of siRNA, mRNA, ASO, plasmid, and saRNA, such as an siRNA drug selected from the cell ferroptosis target genes SLC7A11 or Nrf2.

[0036] According to an embodiment of the present invention, the drug can be an anticancer drug.

[0037] According to an embodiment of the present invention, the drug-loaded lipid nanoparticle composition may further comprise a second drug component, for example, a divalent iron-containing substance, such as ferrocene.

[0038] The present invention also provides a method for preparing the drug-loaded lipid nanoparticle composition, comprising the following steps: loading the drug onto the blank lipid nanoparticles to obtain the drug-loaded lipid nanoparticle composition.

[0039] According to an embodiment of the present invention, the preparation method comprises the following steps:

[0040] (1) Dissolve the drug in a sodium citrate aqueous solution to obtain a drug solution;

[0041] (2) Mix the solution of the blank lipid nanoparticles with sodium citrate buffer to obtain a liposome solution;

[0042] (3) Add the drug solution to the liposome solution for loading to obtain the drug-loaded lipid nanoparticle composition.

[0043] According to an embodiment of the present invention, in step (3), the volume ratio of the drug solution to the liposome solution is 1:(0.5 - 5), for example 1:(0.8 - 3), and preferably 1:1.

[0044] According to an embodiment of the present invention, when the drug-loaded lipid nanoparticle composition further includes a second drug component, the second drug component can be dissolved in the liposome solution and / or the drug solution.

[0045] The present invention also provides the application of the drug-loaded lipid nanoparticle composition in the preparation of anti-cancer drugs.

[0046] According to an embodiment of the present invention, the cancer is breast cancer.

[0047] The present invention also provides a method for treating cancer, including administering a prophylactically or therapeutically effective amount of the drug-loaded lipid nanoparticle composition to a patient.

[0048] According to an embodiment of the present invention, the patient includes mammals, preferably humans.

[0049] The present invention also provides the drug-loaded lipid nanoparticle composition for treating cancer.

[0050] The beneficial effects achieved by the present invention are as follows:

[0051] 1. The present invention designed and synthesized a compound shown in formula (I), which is a novel imidazole ionizable lipid;

[0052] 2. The blank lipid nanoparticles prepared based on the above imidazole ionizable lipid can be used as a drug carrier. The nanoparticles have a uniform particle size, a high drug loading capacity, a suitable pKa and low toxicity, can be ionized in lysosomes, and have excellent endosomal escape effects. Compared with the marketed ionizable lipid MC3 or the commercial transfection reagent Lipo2000, it has higher in vitro and in vivo transfection efficiencies.

[0053] 3. The drug-loaded lipid nanoparticle composition obtained by co-loading ferrous ions and nucleic acid drugs based on the above blank lipid nanoparticles achieves the effect of treating tumors through ferroptosis of cells. Due to the generation of lipid peroxides and the accumulation of reactive oxygen species (ROS) in tumor cells, the drug-loaded lipid nanoparticle composition constructed by the present invention has low toxicity to normal cells while causing cytotoxicity to tumor cells, and therefore has high-efficiency and low-toxic tumor treatment activity. Description of the Drawings

[0054] Figure 1 : Hydrogen NMR spectrum of compound A8-D3-I3;

[0055] Figure 2 : Carbon NMR spectrum of compound A8-D3-I3;

[0056] Figure 3 : Hydrogen NMR spectrum of compound C12-D3-I3;

[0057] Figure 4 : Carbon NMR spectrum of compound C12-D3-I3;

[0058] Figure 5 : Hydrogen NMR spectrum of compound O12-D3-I3;

[0059] Figure 6 : Carbon NMR spectrum of compound O12-D3-I3;

[0060] Figure 7 : Histogram of cytotoxicity test of lipid nanoparticles prepared from compounds in Examples 1-3;

[0061] Figure 8 : Diagram of pKa determination of lipid nanoparticles prepared from compounds in Examples 1-3;

[0062] Figure 9 : Flow cytometry diagram of Cy5-siNC@LNP cell uptake ability;

[0063] Figure 10 : Histogram of fluorescence intensity of Cy5-siNC@LNP cell uptake;

[0064] Figure 11 : Histogram of in vitro knockdown activity test;

[0065] Figure 12 : Histogram of cytotoxicity test of Fc-siSL@LNP and Fc-siNr@LNP on Hep G2 tumor cells;

[0066] Figure 13 : Histogram of cytotoxicity test of Fc-siSL@LNP and Fc-siNr@LNP on BEAS 2B cells;

[0067] Figure 14 : Histogram of cytotoxicity test of Fc-siSL@LNP and Fc-siNr@LNP on 293T cells;

[0068] Figure 15 : Histogram of cytotoxicity test of Fc-siSL@LNP and Fc-siNr@LNP on LO2 cells;

[0069] Figure 16 : Line graph for monitoring the body weight of tumor-bearing mice;

[0070] Figure 17 : Line graph of tumor volume over time;

[0071] Figure 18 : Bar graph of the final tumor volume;

[0072] Figure 19 : Tumor comparison chart.

[0073] Term Definitions and Explanations

[0074] Unless otherwise specified, the definitions of groups and terms recited in the specification and claims of this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions recited in tables, definitions of specific compounds in examples, etc., can be combined and combined with each other arbitrarily. The group definitions and compound structures after such combination and combination should be understood to be within the scope recited in the specification and / or claims of this application.

[0075] Unless otherwise specified, the numerical ranges recited in this specification and claims are equivalent to at least recording each specific integer value therein. For example, the numerical range "0 - 10" is equivalent to recording each integer value in the numerical range "0 - 10", namely 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0076] The term "C 1-10 alkyl" should be understood to mean a straight-chain and branched-chain monovalent alkyl group having 1 to 10 carbon atoms, and "C 1-6 alkyl" means a straight-chain and branched-chain alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc. or their isomers.

[0077] The term "C 1-6 alkyloxy" should be understood as "C 1-6 alkyl - O -", and C 1-10 alkyl is as defined above.

[0078] The term "C 2-6"Alkenyl" should be understood to mean a straight-chain or branched monovalent hydrocarbon group containing one or more double bonds and having 2 to 6 carbon atoms, for example, having 2, 3, 4, 5 or 6 carbon atoms, having 2 or 3 carbon atoms (i.e., C 2-3 alkenyl). It should be understood that in the case where the alkenyl contains more than one double bond, the double bonds may be separated or conjugated to each other. The alkenyl is, for example, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, pent-4-enyl, (E)-pent-3-enyl, (Z)-pent-3-enyl, (E)-pent-2-enyl, (Z)-pent-2-enyl, (E)-pent-1-enyl, (Z)-pent-1-enyl, hex-5-enyl, (E)-hex-4-enyl, (Z)-hex-4-enyl, (E)-hex-3-enyl, (Z)-hex-3-enyl, (E)-hex-2-enyl, (Z)-hex-2-enyl, (E)-hex-1-enyl, (Z)-hex-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl, (Z)-1-methylprop-1-enyl, 3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl, 3-methylbut-2-enyl, (E)-2-methylbut-2-enyl, (Z)-2-methylbut-2-enyl, (E)-1-methylbut-2-enyl, (Z)-1-methylbut-2-enyl, (E)-3-methylbut-1-enyl, (Z)-3-methylbut-1-enyl, (E)-2-methylbut-1-enyl, (Z)-2-methylbut-1-enyl, (E)-1-methylbut-1-enyl, (Z)-1-methylbut-1-enyl, 1,1-dimethylprop-2-enyl, 1-ethylprop-1-enyl, 1-propylvinyl, 1-isopropylvinyl.

[0079] The term "C 3-8 cycloalkyl" should be understood to mean a saturated monovalent monocyclic or bicyclic (such as fused rings, bridged rings, spiro rings) hydrocarbon ring having 3 to 8 carbon atoms, for example having 3, 4, 5, 6, 7 or 8 carbon atoms. The C 3-8 cycloalkyl can be a monocyclic hydrocarbon group, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl, or a bicyclic hydrocarbon group such as bicyclo[2.1.1]hexyl.

[0080] The term "C 6-10 aryl" should be understood to preferably mean a monovalent aromatic or partially aromatic monocyclic or bicyclic having 6, 7, 8, 9 or 10 carbon atoms, especially a ring having 6 carbon atoms ("C 6"Aryl"), such as phenyl; or biphenyl, or a ring having 9 carbon atoms ("C 9 "Aryl"), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 "Aryl"), such as tetrahydronaphthyl, dihydronaphthyl or naphthyl.

[0081] The term "5- to 10-membered heteroaryl" is understood to include a monovalent monocyclic or bicyclic aromatic ring system having 5, 6, 7, 8, 9 or 10 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and containing 1 to 5, preferably 1 to 3 heteroatoms each independently selected from N, O and S and, additionally in each case, may be benzo-fused. "Heteroaryl" also refers to a group in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic or heterocyclic rings, where the attached radical or point is on the heteroaromatic ring.

[0082] The term "3- to 8-membered heterocyclic group" refers to a saturated or unsaturated non-aromatic ring or ring system, for example, which is a 4-, 5-, 6- or 7-membered monocyclic, 7- or 8-membered bicyclic (such as fused, bridged, spiro) ring system and contains at least one, for example 1, 2, 3, 4, 5 or more heteroatoms selected from O, S and N, where N and S may also optionally be oxidized to various oxidation states to form N-oxides, -S(O)- or -S(O) 2 - state. The heterocyclic group may include fused or bridged rings as well as spiro rings. In particular, the heterocyclic group may include but is not limited to: 4-membered rings such as azetidinyl, oxetanyl; 5-membered rings such as tetrahydrofuranyl, dioxolanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or 6-membered rings such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or 7-membered rings such as diazepanyl.

[0083] The term "sub*yl" refers to a divalent organic group that is attached to the main body by two single bonds, such as "sub-C 1-6 "Alkyl" refers to the divalent group formed by eliminating two single bonds from "C 1-6 "Alkane". Detailed Description of the Invention

[0084] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only illustrative of and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0085] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods.

[0086] Example 1: Synthesis of Compound A8-D3-I3

[0087]

[0088] Add A8 (1.21 g, 5.0 mmol) and DIPEA (2.47 g, 19.2 mmol) to 15 mL of anhydrous DMF, stir at room temperature for 2 h, then add D3B (0.91 g, 3.84 mmol), and continue to stir the mixture at room temperature overnight. After the reaction is completed as detected by TLC, wash with saturated NaCl three times, extract the organic phase with DCM, and separate by column chromatography to obtain a colorless transparent liquid A8-D3B (0.74 g) with a yield of 48.5%.

[0089] Under ice bath conditions, slowly add TFA (4.5 mL) to the dichloromethane solution of A8-D3B (1.00 g, 2.5 mmol), stir the reaction for 1 h. After the reaction is completed as detected by TLC, remove the solvent by distillation under reduced pressure, wash with saturated NaHCO 3 three times, and extract the organic phase with EA. Distill under reduced pressure to obtain a pale yellow transparent liquid A8-D3 (700 mg) with a yield of 93.5%.

[0090] Add I3 (1.00 g, 6.0 mmol) to a 20 mL solution of 1,4-dioxane and water (1:1), slowly add TEA (1.95 g, 18.0 mmol), and then slowly dropwise add a 1,4-dioxane solution of (Boc) 2 O (3.50 g, 16.0 mmol) under ice bath conditions, and then react at room temperature overnight. After the reaction is completed as detected by TLC, wash with hydrochloric acid three times, extract the organic phase with EA, and distill under reduced pressure to obtain a white crystalline solid I3-2B2 (2.29 g) with a yield of 100%.

[0091] Under ice bath conditions, successively add I3-2B2 (400 mg, 1.1 mmol), 15 mL of DCM, PyBOP (586 mg, 1.1 mmol), and DIPEA (581 mg, 4.5 mmol) to a round-bottom flask, stir in the ice bath for 20 min, then add A8-D3 (538 mg, 1.4 mmol), and stir at room temperature for 18 h. After the reaction is completed as detected by TLC, wash with saturated NaHCO 3 three times, and extract the organic phase with DCM. After column chromatography, a pale yellow transparent liquid A8-D3-I3-2B2 (400 mg) is obtained with a yield of 55.9%.

[0092] Under ice bath conditions, dissolve A8-D3-I3-2B2 (300 mg, 0.6 mmol) in dichloromethane, slowly add 2 mL of TFA. After the reaction is completed as detected by TLC, distill under reduced pressure, saturated NaHCO3 Wash three times, extract the organic phase with DCM, and obtain pale yellow viscous liquid A8-D3-I3 (100 mg) after column chromatography, with a yield of 48.8%.

[0093] Compound A8-D3-I3 is a pale yellow viscous liquid, MS spectrum (ESI) m / z: C 25 H 49 N 5 O (M + ), theoretical calculated value: 436.39; measured value: 436.40 [M+H] + .

[0094] The hydrogen spectrum of compound A8-D3-I3 is as Figure 1 shown. After measurement, the 1 H NMR of compound A8-D3-I3 is: (600 MHz, CDCl 3 ) δ 7.97 (t, J = 5.3 Hz, 1H), 7.53 (s, 1H), 6.81 (s, 1H), 3.64–3.45 (m, 1H), 3.26 (q, J = 6.4 Hz, 2H), 3.09–2.80 (m, 2H), 2.42 (dt, J = 39.5, 7.3 Hz, 6H), 1.71–1.53 (m, 2H), 1.45–1.36 (m, 4H), 1.25 (m, 20H), 0.85 (t, J = 7.0 Hz, 6H);

[0095] Figure 2 shown 13 C NMR spectrum, 13 C NMR is: (150 MHz, CDCl 3 ) δ 173.78, 134.13, 54.26, 52.95, 51.11, 37.23, 30.83, 28.55, 28.31, 26.58, 25.56, 21.64, 13.09.

[0096] Example 2: Synthesis of compound C12-D3-I3

[0097]

[0098] Add D3B (1.50 g, 8.6 mmol) and C12 (3.49 g, 18.9 mmol) to 15 mL of ethanol, stir at room temperature for 36 h. After TLC detection shows the reaction is complete, wash three times with saturated NaCl, and separate by column chromatography to obtain white blocky solid C12-D3B (2.95 g), with a yield of 63.1%.

[0099] Under ice bath conditions, TFA (5 mL) was slowly added to a dichloromethane solution of C12-D3B (1.00 g, 2.3 mmol), and the mixture was stirred for 1 h. After the reaction was completed as detected by TLC, the solvent was removed by distillation under reduced pressure, and the mixture was washed three times with saturated NaHCO 3 and the organic phase was extracted with DCM. After distillation under reduced pressure, a pale yellow transparent liquid C12-D3 was obtained with a yield of 80.2%.

[0100] Under ice bath conditions, I3-2B2 (1.12 g, 3.2 mmol), DCM (15 mL), HATU (1.20 g, 3.2 mmol), and DIPEA (812 mg, 6.3 mmol) were successively added to a round-bottom flask, and the mixture was stirred in an ice bath for 20 min. Then C12-D3 was added, and the mixture was stirred at room temperature for 18 h. After the reaction was completed as detected by TLC, the mixture was washed three times with saturated NaHCO 3 and the organic phase was extracted with DCM. After column chromatography, a pale yellow transparent liquid C12-D3-I3-2B2 (3.49 g, 18.9 mmol) was obtained with a yield of 36.4%.

[0101] Under ice bath conditions, C12-D3-I3-2B2 (390 mg, 0.6 mmol) was dissolved in dichloromethane, and TFA (3 mL) was slowly added dropwise. The reaction was carried out for 1 h. After the reaction was completed as detected by TLC, the mixture was distilled under reduced pressure, washed three times with saturated NaHCO 3 and the organic phase was extracted with DCM. After column chromatography, a pale yellow viscous liquid C12-D3-I3 (160 mg) was obtained with a yield of 58.1%.

[0102] Compound C12-D3-I3 is a pale yellow viscous liquid. MS spectrum (MALDI) m / z: C 33 H 65 N 5 O 3 (M + ), theoretical calculated value: 580.509; measured value: 580.378 [M+H] + .

[0103] The 1H NMR spectrum of compound C12-D3-I3 is as Figure 3 shown. As determined, the 11H NMR was as follows: (600 MHz, CDCl3) δ 7.51–7.43 (m, 1H), 6.81–6.67 (m, 1H), 4.58 (s, 2H), 3.77–3.65 (m, 2H), 3.65–3.53 (m, 2H), 2.99–2.83 (m, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.56–2.38 (m, 4H), 2.24 (dd, J = 12.2, 3.4 Hz, 1H), 1.72–1.56 (m, 2H), 1.47–1.34 (m, 6H), 1.33–1.17 (m, 30H), 0.87 (t, J = 7.0 Hz, 6H);

[0104] Figure 4 as shown 13 13C NMR spectrum 13 13C NMR was as follows: (150 MHz, CDCl3) δ 173.47, 134.01, 133.93, 113.07, 69.41, 66.95, 62.75, 60.35, 55.20, 54.13, 37.59, 34.47, 34.28, 30.90, 28.82, 28.62, 28.33, 25.55, 24.84, 21.68, 13.11.

[0105] Example 3: Synthesis of Compound O12-D3-I3

[0106]

[0107] TEA (1.45 g, 14.4 mmol) was slowly added dropwise to a solution of D3B (1.00 g, 5.7 mmol) in IPA (15 mL). After reacting for 1 h, O12 (3.45 g, 14.4 mmol) was added, and the reaction temperature was raised to 90 °C for reflux reaction for 5 h. After completion of the reaction detected by TLC, it was washed with saturated NaCl three times, the organic phase was extracted with DCM, and column chromatography was carried out to obtain a colorless transparent viscous liquid O12-D3B (0.75 g), with a yield of 20.1%.

[0108] Under ice bath conditions, TFA (5 mL) was slowly added to a dichloromethane solution of O12-D3B (1.00 g, 1.5 mmol), and the mixture was stirred and reacted for 1 h. After completion of the reaction detected by TLC, the solvent was removed by distillation under reduced pressure, and it was washed with saturated NaHCO 3 three times, and the organic phase was extracted with EA, and distillation under reduced pressure gave a pale yellow transparent liquid O12-D3 (1.18 g), with a yield of 100%.

[0109] Under ice bath conditions, I3-2B2 (639 mg, 1.8 mmol), DCM (15 mL), PyBOP (468 mg, 0.9 mmol), and DIPEA (464 mg, 3.6 mmol) were successively added to a round-bottom flask, and the mixture was stirred in an ice bath for 20 min. Then, O12-D3 (500 mg, 0.9 mmol) was added, and the mixture was stirred at room temperature for 18 h. After the reaction was completed as detected by TLC, it was washed three times with saturated NaHCO 3 and the organic phase was extracted with DCM. After column chromatography, a pale yellow transparent liquid O12-D3-I3-2B2 (390 mg) was obtained with a yield of 53.5%.

[0110] Under ice bath conditions, O12-D3-I3-2B2 (360 mg, 0.4 mmol) was dissolved in dichloromethane, and TFA (3 mL) was slowly added dropwise. The reaction was carried out for 1 h. After the reaction was completed as detected by TLC, it was distilled under reduced pressure and washed three times with saturated NaHCO 3 and the organic phase was extracted with DCM. After column chromatography, a pale yellow viscous liquid O12-D3-I3 (150 mg) was obtained with a yield of 53.8%.

[0111] Compound O12-D3-I3 is a pale yellow viscous liquid. MS spectrum (MALDI) m / z: C 39 H 73 N 5 O 5 (M + ), theoretical calculated value: 692.561; measured value: 692.400 [M+H] + .

[0112] The hydrogen spectrum of compound O12-D3-I3 is as Figure 5 shown. It was determined that the 1 1H NMR of compound O12-D3-I3 is: (600 MHz, CDCl 3 ) δ 7.76 (s, 1H), 7.56 (s, 1H), 6.82 (s, 1H), 4.75 (s, 3H), 4.02 (t, J = 6.8 Hz, 4H), 3.20 (ddd, J = 43.0, 13.3, 6.5 Hz, 2H), 3.05–2.90 (m, 2H), 2.71 (d, J = 7.1 Hz, 4H), 2.39 (dt, J = 19.6, 6.9 Hz, 6H), 1.58 (dq, J = 13.1, 6.6 Hz, 6H), 1.37–1.17 (m, 37H), 0.85 (t, J = 7.0 Hz, 6H);

[0113] Figure 6 as shown in the 13 13C NMR spectrum shown, 13 13C NMR is:13 C NMR (151 MHz, CDCl 3 ) δ 173.29, 171.93, 138.26, 134.09, 131.38, 63.83, 54.01, 50.14, 48.11, 36.41, 31.49, 30.91, 28.68, 28.65, 28.63, 28.60, 28.54, 28.34, 28.29, 27.60, 25.65, 24.93, 24.93, 21.68, 13.11。

[0114] Example 4: Preparation of Ionizable Lipid-Based Blank Lipid Nanoparticles and Drug-Loaded Lipid Nanoparticles

[0115] 1. Experimental Method

[0116] The compounds prepared in Examples 1-3 were used as ionizable lipids to prepare blank lipid nanoparticles and drug-loaded lipid nanoparticles. Blank lipid nanoparticles were prepared from lipid nanoparticle raw materials (ionizable lipid, DSPC, cholesterol, DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5) by the thin film hydration method or the ethanol injection method, and the mass ratio of blank lipid nanoparticles to siRNA was 15:1. Experimental methods: i) Thin film hydration method: The lipid nanoparticle raw materials were dissolved in a round-bottom flask containing 1 mL of chloroform according to the ratio, and ultrasonicated at 25 °C and 150 W to completely dissolve to obtain a lipid stock solution. The chloroform in the round-bottom flask was evaporated to dryness at 45 °C using a rotary evaporator to form a honeycomb-like film on the inner wall of the flask. 1 mL of drug-containing buffer solution (such as PBS solution of siRNA) was added to the flask, and rotated for hydration for 35 min (1 mL of buffer solution (such as PBS solution) was added to the flask and rotated for hydration to obtain blank lipid nanoparticles), and the resulting suspension was ultrasonicated for 2 min using a cell disruption ultrasonic device, and then filtered through a 0.22 μm microporous filter membrane to obtain drug-loaded lipid nanoparticles. ii) Ethanol injection method: The lipid nanoparticle raw materials were dissolved in ethanol according to the ratio to obtain a lipid stock solution. Three times the volume of ethanol of drug-containing buffer solution (such as siRNA citrate buffer solution (0.05 M, pH = 4.0)) was placed in a penicillin bottle, and the lipid stock solution was rapidly injected into the drug-containing buffer solution under stirring (the lipid stock solution was rapidly injected into the buffer solution under stirring to obtain blank lipid nanoparticles) to obtain a drug-loaded lipid nanoparticle solution. Then it was incubated at 50 °C for 20 min, and then the drug-loaded lipid nanoparticle solution was dialyzed in 1×PBS for at least 2 hours to obtain drug-loaded lipid nanoparticles. After preparation, samples were taken to detect the encapsulation efficiency, average particle size, PDI and Zeta potential.

[0117] 2. Experimental Results

[0118] The characterization data of blank lipid nanoparticles prepared with different ionizable lipids after loading the negative control siNC are shown in the following table. After loading siNC, the particle sizes of the three blank lipids increased slightly, and the encapsulation efficiency was about 90%. The above results indicate that under the same preparation conditions, the particle sizes of siNC@LNP prepared with the three ionizable lipids are uniform, and the ionizable lipids have a high encapsulation effect on siRNA.

[0119]

[0120] Example 5: In vitro toxicity test of drug-loaded lipid nanoparticles

[0121] 1. Experimental method

[0122] The cytotoxic effect of siNC@LNP on cells was detected using the CCK-8 method. Hep G2 cells in the logarithmic growth phase were collected, and the cell concentration was adjusted and inoculated into 96-well plates so that the concentration of the cells to be tested was 8000 / well. After incubating at 5% CO 2 , 37 °C for 24 hours, the medium in the 96-well plates was replaced with medium without antibiotics, and siNC@LNP with final concentrations of 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM of siRNA was added. After incubating at 37 °C for 24 h, the medium was discarded, 90 μL of PBS and 10 μL of CCK-8 were added to each well, and incubation was continued for 1 hour. The absorbance of each well at 450 nm was measured using a microplate reader.

[0123] 2. Experimental results

[0124] The experimental results are as Figure 7 shown. The results indicate that when the administration concentration of siNC@LNP reached 800 μM, no obvious toxicity was observed in all lipid nanoparticles.

[0125] Example 6: pKa determination of blank lipid nanoparticles

[0126] 1. Experimental method

[0127] To determine the pKa of blank lipid nanoparticles (LNP) prepared with three imidazole ionizable lipids, the prepared LNP was diluted to 100 μM with a buffer containing 130 mM NaCl, 10 mM ammonium acetate, 10 mM Hepes, and 10 mM MES. Then the solution was divided into 19 equal parts, and the pH of each solution was adjusted in the range of 2.5 to 11 using NaOH or HCl. 2-(p-Tolyl)-6-naphthalenesulfonic acid (TNS) was dissolved in distilled water and added to the pH-adjusted LNP solution to a final concentration of 1 μM. The fluorescence intensity of the solutions at different pH values was measured using a microplate reader at room temperature with an excitation wavelength of 321 nm and an emission wavelength of 445 nm. The pKa was defined as the pH at which the fluorescence intensity increased to half of the maximum fluorescence intensity.

[0128] 2. Experimental Results

[0129] The pKa values of LNPs were determined by the TNS method. As Figure 8 shown, the pKa values of the three blank lipid nanoparticles were 6.75, 6.39, and 8.18, respectively. The pKa value of the blank lipid nanoparticles must reach or exceed 5.5, and the pKa range of 6.2 - 6.9 has been proven to be the optimal range for protein expression after nucleic acid delivery. The blank lipid nanoparticles prepared with C12-D3-I3 and O12-D3-I3 ionizable lipids meet the ideal pKa required for siRNA transport.

[0130] Example 7: Determination of the Cellular Uptake Ability of Cy5-siNC@LNP

[0131] 1. Experimental Method

[0132] Hep G2 cells were seeded into six-well plates one day before transfection. The next day, the adherent cells were treated with cy5-siNC@LNP and siNC@LNP, and cy5-siNC@MC3 and cy5-siNC@Lipo 2000 were used as positive controls. After 4 hours, the cells were digested with trypsin (0.25%) and washed three times with PBS, and then resuspended in 300 μL of 1×PBS. The measurement was performed by flow cytometry, and the cellular uptake ability was analyzed using the software Flowjo v10.

[0133] 2. Experimental Results

[0134] The experimental results are as Figure 9 , Figure 10 shown. The cellular uptake ability of the drug-loaded lipid nanoparticles prepared with the three imidazole ionizable lipids was higher than that of the positive controls MC3 and Lipo2000. Among them, the drug-loaded lipid nanoparticles prepared with O12-D3-I3 had the strongest cellular delivery ability.

[0135] Example 8: In Vitro Knockdown Activity Test

[0136] 1. Experimental Method

[0137] 293T-Luc stably transfected with firefly luciferase was seeded into 96-well plates. The next day, the siLuc targeting firefly luciferase was encapsulated with three newly prepared blank lipid nanoparticles, and MC3 was used as a positive control. Samples with a final concentration of 50 nM of siLuc were added to the corresponding wells and incubated for 4 h. The complete medium was replaced and the cells were cultured for another 20 h. Then, the cells were collected using cell lysis buffer, and luciferin was added to the lysed cells. The chemiluminescence intensity was immediately determined using a microplate reader.

[0138] 2. Experimental Results

[0139] The experimental results are as follows Figure 11 shown. Among them, A8-D3-I3 is denoted as A-D-I, C12-D3-I3 is denoted as C-D-I, O12-D3-I3 is denoted as O-D-I. The knockdown activity of O12-D3-I3 in vitro is better than that of the positive control MC3. Therefore, O12-D3-I3 is used for subsequent activity tests.

[0140] Example 9: Cytotoxicity Test of Fc-siSL@LNP and Fc-siNr@LNP on Tumor Cells

[0141] 1. Experimental Method

[0142] Drug-loaded lipid nanoparticles Fc-siSL@LNP and Fc-siNr@LNP are prepared using O-D-I to load siRNA of the ferroptosis target genes SLC7A11 or Nrf2 of cells and ferrocene (Fc) for evaluating the inhibitory effect on tumor cells. Hep G2 cells in the logarithmic growth phase are collected, the cell suspension concentration is adjusted, and the cells are seeded in a 96-well plate at a cell density of 8000 / well. After incubation at 5% CO 2 , 37°C for 24 hours, the medium in the 96-well plate is replaced with medium without antibiotics, and Fc@LNP, Fc-siSL@LNP or Fc-siNr@LNP with a final concentration of siRNA of 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM are added. After incubation at 37°C for 24 hours, the medium is discarded, 90 μL of PBS and 10 μL of CCK-8 are added to each well, and incubation is continued for 30 min. The absorbance of each well at 450 nm is detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0143] 2. Experimental Results

[0144] The experimental results are as follows Figure 12 shown. The results show that Fc-siSL@LNP and Fc-siNr@LNP play a role in inhibiting the growth of tumor cells by inducing ferroptosis of cells. In addition, co-loading ferrous ion ferrocene and ferroptosis target gene siSLC7A11 or siNrf2 has a stronger effect on inducing ferroptosis of cells.

[0145] Example 10: Cytotoxicity Test of Fc-siSL@LNP and Fc-siNr@LNP on Normal Cells

[0146] 1. Experimental Method

[0147] The siRNAs targeting the ferroptosis-related genes SLC7A11 or Nrf2 were loaded into O-D-I and used to prepare drug-loaded lipid nanoparticles Fc-siSL@LNP and Fc-siNr@LNP with ferrocene (Fc) for the cytotoxicity evaluation of normal cells. BEAS 2B, 293T, and LO2 cells in the logarithmic growth phase were collected, the cell suspension concentration was adjusted, and the cells were seeded in 96-well plates at a cell density of 8000 cells / well. After incubation at 37 °C in 5% CO 2 , for 24 hours, the medium in the 96-well plates was replaced with medium without antibiotics, and Fc-siSL@LNP or Fc-siNr@LNP with final siRNA concentrations of 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM were added. After incubation at 37 °C for 24 hours, the medium was discarded, 90 μL of PBS and 10 μL of CCK-8 were added to each well, and incubation was continued for 30 min. The absorbance of each well at 450 nm was measured using a microplate reader.

[0148] 2. Experimental results

[0149] The results are shown in Figure 13 , 14 and 15. The experimental results showed that Fc-siSL@LNP and Fc-siNr@LNP showed no significant toxicity to normal cells at 800 nM. Since ferroptosis requires the participation of reactive oxygen species and the accumulation of lipid peroxides, tumor cells are more prone to ferroptosis due to the presence of excessive reactive oxygen species, enabling Fc-siSL@LNP or Fc-siNr@LNP to specifically act on tumor cells with low toxicity to normal cells.

[0150] Example 11: Anticancer effect of drug-loaded lipid nanoparticle formulations in tumor-bearing mice

[0151] 1. Experimental method

[0152] To establish a xenograft tumor model, breast cancer cells MCF-7 were washed twice with PBS and resuspended in PBS to a cell density of 1.5×10 7 / mL, and 200 μL of the tumor cell suspension was injected into the right axilla of the mice. When the tumors grew to 100 - 200 mm 3 , the mice were randomly divided into 4 groups: (1) Control group, (2) Fc@LNP group, (3) Fc-siSL@LNP group, and (4) Fc-siNr@LNP group. Administration was carried out on days 0, 3, 6, 9, and 12 after grouping. Body weight and tumor volume were recorded during the treatment process. The tumor-bearing mice were sacrificed on day 15, and the tumor tissues were isolated.

[0153] 2. Experimental results

[0154] During the treatment process, the body weight of the mice was continuously monitored. As Figure 16 shown, the body weight of the mice in the Control group showed a downward trend after the 6th day, and there were no obvious changes in the body weight of the mice in the other groups. The tumor volume changed over time, the final tumor volume, and the tumor images of each group are shown in Figure 16 , 17 and Figure 18. Compared with the control group, the other three groups all had varying degrees of in vivo tumor suppression ability. Among them, the Fc-siSL@LNP group and the Fc-siNr@LNP group had a significant tumor suppression effect compared with the Fc@LNP group, and the tumor size did not show obvious growth during the experiment. Therefore, the use of imidazole ionizable lipid O12-D3-I3 co-loaded with divalent iron and ferroptosis-related target siRNA can effectively induce ferroptosis in tumor cells and is non-toxic to normal cells at the administered concentration.

[0155] In summary, the present invention designed and synthesized an imidazole ionizable lipid with a novel parent nucleus structure. The blank or drug-loaded liposomes prepared using O12-D3-I3 had excellent in vitro and in vivo transfection activities, which were superior to the commercially available positive control MC3. By co-loading ferrous ion ferrocene and ferroptosis target gene siSLC7A11 or siNrf2, the prepared Fc-siSL@LNP group and Fc-siNr@LNP had excellent in vitro and in vivo tumor suppression activities and had no significant toxicity to normal cells, demonstrating the excellent transfection ability of O12-D3-I3.

[0156] The above is an exemplary description of the implementation manner of the technical solution of the present invention. It should be understood that the protection scope of the present invention is not limited to the above implementation manner. Any modifications, equivalent replacements, improvements, etc. made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the claims of this application.

Claims

1. A compound represented by formula (I): Wherein, M 1 and M 2 are the same and are each independently selected from -CH 2 - and -CH(OH)-; G 1 and G 2 are the same and are each independently selected from -CH 2 -, -CH(OH)-; E 1 and E 2 are the same and are each independently selected from -CH 2 -, -OC(=O)-, -C(=O)O-; L 1 Selected from: R 1 selected from -NH 2 ; J 1 Selected from -CH 2 -; n is selected from the numbers 5 - 22.

2. The compound according to claim 1, characterized in that, the compound represented by formula (I) is selected from the compounds having the following structures:

3. A blank lipid nanoparticle, comprising the compound according to claim 1 or 2, phospholipid, cholesterol and PEG lipid; the phospholipid is selected from at least one of DSPC, DPPC, POPC, DOPE and DEPC; the PEG lipid is selected from at least one of DSPE - PEG 2000, MG - PEG 2000, DPPE - PEG 2000, DMA - PEG 2000 and DMG - PEG 2000.

4. The blank lipid nanoparticle according to claim 3, characterized in that, the molar ratio of the compound according to claim 1 or 2, phospholipid, cholesterol, PEG lipid is 50:10:38.5:1.

5.

5. The preparation method of the blank lipid nanoparticle according to claim 3 or 4, comprising the following steps: 1) Dissolve the compound represented by formula (I), cholesterol, PEG lipid and phospholipid in an organic solvent to obtain a lipid stock solution; 2) Inject the lipid stock solution into a sodium citrate buffer solution to obtain the blank lipid nanoparticle.

6. The application of the blank lipid nanoparticle according to claim 3 or 4 as a drug carrier.

7. A drug - loaded lipid nanoparticle composition, comprising the blank lipid nanoparticle according to claim 3 or 4 and a drug.

8. The composition according to claim 7, characterized in that, the drug is a nucleic acid drug, and the nucleic acid molecule is selected from at least one of siRNA, mRNA, ASO, plasmid and saRNA.

9. The composition according to claim 7, characterized in that, the drug is selected from siRNA drugs targeting the ferroptosis - related genes SLC7A11 or Nrf2.

10. The composition according to claim 7, characterized in that, the drug is an anti - tumor drug.

11. The composition according to claim 7, characterized in that, the drug - loaded lipid nanoparticle composition further comprises a second drug component.

12. The composition according to claim 11, characterized in that, the second drug component is a divalent iron - containing substance.

13. The composition according to claim 11, characterized in that, the second drug component is ferrocene.

14. The preparation method of the drug - loaded lipid nanoparticle composition according to claim 7, comprising the following steps: Load the drug onto the blank lipid nanoparticle to obtain the drug - loaded lipid nanoparticle composition.

15. The preparation method according to claim 14, characterized in that, the preparation method comprises the following steps: (1) Dissolve the drug in a sodium citrate aqueous solution to obtain a drug solution; (2) Mix the solution of the blank lipid nanoparticle with a sodium citrate buffer solution to obtain a liposome solution; (3) Add the drug solution to the liposome solution for loading to obtain the drug - loaded lipid nanoparticle composition.

16. The application of the drug - loaded lipid nanoparticle composition according to claim 7 in the preparation of anti - cancer drugs.

17. The application according to claim 16, wherein, the cancer is breast cancer.

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