Preparation and application of novel ionizable lipid molecule

Abstract

The invention provides preparation and application of a novel ionizable lipid molecule, the molecule comprises a disulfide bond and an isomannitol structure, and lipid nanoparticles (LNPs) prepared by using the ionizable lipid molecule have excellent encapsulation efficiency and can significantly improve the efficiency of delivering drugs to cells and animals. The lipid nanoparticles prepared from the ionizable lipid molecules containing the disulfide bond and the isomannitol structure can improve the stability, compatibility and safety of the lipid nanoparticles in a living body, and the lipid nanoparticles can regulate and control the activity of drugs, increase the stability of the drugs and improve the availability of organisms to the drugs; and an efficient delivery way is provided for the nucleic acid medicine, so that the development of the nucleic acid medicine is promoted.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the preparation and application of a novel ionizable lipid molecule, and in particular to the preparation and application of a novel ionizable lipid molecule containing a disulfide bond and an isomannide structure. Background Art

[0002] mRNA vaccines guide the body to synthesize specific antigen proteins, activate the immune system to produce specific immune responses, improve the body's immunity to diseases, and achieve the purpose of preventing infection or disease. This technology also shows great potential in fields such as cancer immunotherapy. Compared with other drugs, mRNA has the advantages of high safety, effectiveness, customization, and relatively easy industrial production. However, it also has its own disadvantages. The single strand of mRNA is unstable and easily degraded; most mRNAs carry negative charges and are difficult to penetrate the cell membrane, so the use of mRNA alone is not ideal. Therefore, special modifications or the use of package delivery systems are required to achieve efficient delivery of mRNA, change the biodistribution, cell targeting and uptake mechanism of mRNA cells, and better exert the effect of mRNA drugs. Therefore, the design and development of more efficient and safe mRNA delivery systems has become the key to nucleic acid delivery. Commonly used nucleic acid delivery methods mainly include: lipid nanoparticles, proteins, viral vectors, electroporation, polymer carriers, etc. Lipid nanoparticles (LNPs) are considered to be the most effective mRNA delivery technology at present, and are also the main delivery carriers used by COVID-19 mRNA vaccines.

[0003] LNPs are a lipid-based delivery vehicle that protects nucleic acid payloads from degradation and can deliver nucleic acids into the cytoplasm; when used for vaccination, they can also act as adjuvants themselves. LNPs are a versatile platform that can encapsulate various types of RNA as payloads and administer them through different routes. LNPs are usually composed of four lipid components (phospholipids, cholesterol, auxiliary lipids, and ionizable lipids), of which ionizable cationic liposomes account for about 50% of the entire component, so ionizable cationic liposomes are the core of LNPs. So far, only three cationic liposomes have been approved for marketing: Dlin-MC3-DMA, SM-102, and ALC-0315. Therefore, the development of efficient and safe ionizable cationic liposomes is a key issue in solving the core technology of LNPs.

[0004] CN118063774A discloses a lipoic acid polymer that can be used to assist in nucleic acid delivery, its preparation method and application. The structural formula of the lipoic acid polymer contains multiple disulfide bonds, which can partially or completely replace ionizable lipid molecules. It does not mainly consist of a carbon chain backbone. The increase in disulfide bonds may make the structure of lipid molecules too rigid, affecting their flexibility and interaction with other molecules, and excessive disulfide bonds may change the biological activity of lipid molecules, which may lead to the drug delivery carriers formed by lipid molecules being unable to function properly in vivo; in addition, in some cases, the abnormal accumulation of intracellular disulfides may induce disulfide stress, which is highly toxic to cells. Although this is mainly for proteins, excessive disulfide bonds in lipid molecules may also exacerbate this stress response to a certain extent and cause damage to cells.

[0005] Therefore, there is an urgent need to design a new type of ionizable lipid molecule that can make LNPs have more efficient delivery ability for in vivo and in vitro delivery of nucleic acid drugs, solve the problem of difficult nucleic acid drug delivery, and thus further promote the development of nucleic acid drugs. Summary of the Invention

[0006] To solve the above difficulties, the present invention provides the preparation and application of a new type of ionizable lipid molecule. This molecule contains disulfide bonds and isomannitol structures. Lipid nanoparticles (LNPs) prepared using this ionizable lipid molecule have excellent encapsulation efficiency and can significantly improve the efficiency of delivering drugs to cells and animals. Lipid nanoparticles prepared from ionizable lipid molecules containing disulfide bonds and isomannitol structures can improve their stability, compatibility and safety in vivo. The lipid nanoparticles can not only regulate the activity of drugs, increase drug stability and improve the bioavailability of drugs, but also provide an efficient delivery route for nucleic acid drugs, thus promoting the development of nucleic acid drugs.

[0007] On the one hand, an ionizable lipid molecule has a structure shown in Formula I, or contains the structure shown in Formula I or its derivatives.

[0008]

[0009] Wherein, T is selected from C 3-20 alkyl single chain, or a double chain with the number of carbon atoms greater than or equal to 3. One of the double chains is selected from -(CH 2 ) a C=C(CH 2 ) b C=C(CH 2 ) c , or -(CH 2 ) a OC(=O)(CH 2 )b or a, b, and c are independently selected from integers from 1 to 10; X is selected from -CH 2 OC(=O)NH-, -CH 2 OC(=O)O-, -CH 2 C(=O)O-, -CH 2 C(=O)NH-, -CH 2 C(=O)S-, -CH 2 O-; Y is selected from any substituted or unsubstituted C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, C 3-8 cycloalkenyl, C 3-8 cycloalkynyl, 4- to 8-membered heterocycle; Z is selected from -O(C=O)(CH 2 ) n -, -O(CH 2 ) n -, -O(C=O)O-, -O(C=O)-, -O(C=O)O(CH 2 ) n -; where n is an integer between 1 and 7; A is selected from R1 and R2 are each independently selected from any substituted or unsubstituted C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, C 3-8 cycloalkenyl, C 3-8 cycloalkynyl, phenyl, -(C=O)C 1-3 alkyl; or R1 and R2 combine to form any substituted or unsubstituted 4- to 8-membered heterocycle, pyrimidine ring, purine ring; where the substituent groups are 1 or 2 or 3 or 4 or 5 independent OH, SH, nitro, cyano, amino, C 1-3 hydroxy, C 1-3 alkoxy, -(C=O)OC 1-3 alkyl, C 1-3 alkyl; M is selected from an N atom or a C atom.

[0010] Compared with alkyl backbones without disulfide bonds, the presence of disulfide bonds in LNPs can form crosslinked structures, enhancing the stability of nanoparticles, helping to prevent the dissociation and degradation of nanoparticles, and prolonging their circulation life in vivo. Disulfide bonds are naturally present in living organisms, so LNPs with introduced disulfide bonds usually have good biocompatibility and biosafety. When LNPs enter cells or a specific environment, the disulfide bonds can break to release the encapsulated drugs from the nanoparticles, achieving a controlled release effect.

[0011] Further, the structural formula of Y is as follows:

[0012] The structure of Y is an isomannitol structure, which can make the ionizable lipid molecules more stable. As a polyol, isomannitol has the potential to interact with the phospholipid molecules of liposomes and may enhance the stability of the liposome bilayer membrane through interactions such as hydrogen bonds.

[0013] The ionizable lipid molecules provided by the present invention contain both a disulfide bond structure and an isomannitol structure. The coexistence of these two structures can improve the stability of the ionizable lipid molecules. Because introducing a disulfide bond into the liposome structure can form a more stable liposome skeleton, thereby improving the overall stability of the liposome, and enabling the liposome to release the loaded drug under specific reducing conditions to achieve precise drug delivery. The addition of isomannitol may regulate its permeability by changing the fluidity or permeability of the liposome bilayer membrane. This regulatory effect helps to achieve controlled and sustained release of drugs, and improve the encapsulation efficiency and delivery efficiency of liposomes for genes. Combining the stability of disulfide bonds and the biocompatibility of isomannitol, a more stable liposome structure with higher bioavailability can be constructed.

[0014] Further, T is selected from any one of the structures shown in Formulae T1 - T7:

[0015]

[0016] Further, A is selected from any one of the structures shown in Formulae A1 - A18:

[0017]

[0018] The above different groups A have different transfection effects on the disulfide bond skeleton ionizable lipids. The reason is that different groups A will result in different cationic charges and different degrees of matching with the skeleton, thus leading to different transfection efficiencies of liposomes. The present invention screens and optimizes group A to find group A with good transfection effects and convenient synthesis for preparing ionizable lipid molecules with a disulfide bond skeleton.

[0019] Further, the structural formula of T is as shown in Formula T7; the structural formula of A is selected from any one or more of the structures shown in Formulae A1, A2, A3, A4, A5, A6.

[0020] In some embodiments, the structure of A is preferably A1, A4, A5.

[0021] Further, the structural formula of X is: -CH2C(=O)O-; the structural formula of Z is: -O(C=O)(CH 2 )3 -.

[0022] Further, it includes any one or more of the formulas F1 - F6:

[0023]

[0024]

[0025] On the other hand, a nanoparticle composition includes the ionizable lipid molecule, neutral lipid, steroid, and polymer conjugation described above.

[0026] The nanoparticle composition contains one or more neutral lipids, one or more steroids, and one or more polymer-conjugated lipids; wherein, the molar percentage of the above ionizable lipid molecule is 20 - 100%; the molar percentage of the above steroid is 0 - 80%; the molar percentage of the above neutral lipid is 0 - 40%; the molar percentage of the above polymer-conjugated lipid is 0 - 20%.

[0027] The neutral lipid can provide stability and biocompatibility, and it includes at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin (SM), ceramide, sterol, and its derivatives.

[0028] In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0029] The steroid can increase the fluidity and stability of the liposome membrane and improve the bioavailability of the liposome, and it includes at least one of cholesterol, coprosterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatidine, ursolic acid, α-tocopherol, corticosteroid, and its derivatives.

[0030] In some embodiments, the steroid compound is cholesterol.

[0031] The polymer-conjugated lipid can prevent liposome aggregation, improve its stability, transfection efficiency and biocompatibility, enabling the liposome to achieve long circulation in the blood, thereby prolonging the half-life of the drug in vivo and improving the bioavailability of the drug. The polymer-conjugated lipid comprises at least one of polyethylene glycol-modified phosphatidylethanolamine, polyethylene glycol-modified phosphatidic acid, polyethylene glycol-modified ceramide, polyethylene glycol-modified dialkylamine, polyethylene glycol-modified diacylglycerol, and polyethylene glycol-modified dialkylglycerol.

[0032] In some embodiments, the polymer-conjugated lipid is DMG-PEG2000.

[0033] In yet another aspect, there is provided the use of the nanoparticle composition for preparing a delivery vehicle.

[0034] In some embodiments, the ionizable cationic lipid molecules of F1-F6 and the commercial lipid material SM-102 are respectively formulated into drug delivery vehicles with DSPC, cholesterol, and DMG-PEG2000 and encapsulate mRNA. It is demonstrated by detection that the encapsulation efficiency of the lipid nanoparticle delivery vehicles prepared from the ionizable cationic lipid molecules of F1-F6 for mRNA is higher than that of the lipid nanoparticle delivery vehicles prepared from the commercial lipid material SM-102 for mRNA.

[0035] In yet another aspect, there is provided the use of the ionizable lipid molecule as described above for preparing a preparation for improving the transfection efficiency of a drug, wherein the drug includes a therapeutic or prophylactic agent.

[0036] The therapeutic or prophylactic agent is selected from any one or more of nucleic acid drugs, nucleic acid vaccines, small molecule drugs, polypeptide drugs, protein drugs, and drug active molecules. The nucleic acid drug is selected from any one or more of DNA drugs and RNA drugs. The nucleic acid drug is selected from any one or more of single-stranded DNA, double-stranded DNA, short isoforms, agomir, antagomir, antisense molecules, small interfering RNA (siRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), circular RNA (circRNA), aRNA, lncRNA, and nucleic acid aptamers.

[0037] In yet another aspect, the present invention provides a method for delivering a nucleic acid to a cell and / or an organ using the above-described delivery vehicle.

[0038] The administration routes of the delivery vehicle include, but are not limited to, intravenous, intramuscular, intradermal, subcutaneous, or intranasal administration.

[0039] In some ways, the effects of lipid nanoparticle delivery carriers containing ionizable cationic lipid molecules F1 - F6 and lipid nanoparticle delivery carriers containing commercial lipid material SM-102 on transfecting mRNA into two different cells (lung cancer cells and renal cells) were detected respectively. Experiments proved that the lipid nanoparticle delivery carriers containing F1, F4, and F5 had higher efficiency in transfecting mRNA into the two different cells than the lipid nanoparticle delivery carriers containing commercial lipid material SM-102. F1, F4, and F5 were preferably selected as the ionizable lipid molecules for preparing lipid nanoparticle delivery carriers.

[0040] In some ways, animal experiment transfection experiments were carried out on the lipid nanoparticle delivery carriers containing F1, F4, and F5 with high cell transfection efficiency and the lipid nanoparticle delivery carriers containing commercial lipid material SM-102 to detect the effects of these three lipid nanoparticle delivery carriers on transfecting mRNA into mice. Experiments proved that the lipid nanoparticle delivery carrier containing F1 had better effect on transfecting mRNA into mice than the other three lipid nanoparticle delivery carriers. F1 was preferably selected as the ionizable lipid molecule for preparing lipid nanoparticle delivery carriers.

[0041] In some ways, the ionizable lipid molecule F1 and ionizable lipid molecules without isomannitol structure or without disulfide bond structure were respectively prepared into lipid nanoparticle delivery carriers, and their particle size, polydispersity index, zeta potential, encapsulation efficiency for different mRNAs, and transfection efficiency for different cells were detected respectively. The detection results proved that only when the ionizable lipid molecule contains both disulfide bond structure and isomannitol structure can the stability of nanoparticles be enhanced, the encapsulation efficiency be improved, the dissociation and degradation of nanoparticles be prevented, and the cell transfection efficiency be further improved.

[0042] The present invention has the following beneficial effects:

[0043] (1) A novel ionizable lipid molecule is provided, which contains a disulfide bond and an isomannitol structure. The lipid nanoparticles (LNPs) prepared by using this ionizable lipid molecule have excellent encapsulation efficiency, and the encapsulation efficiency is higher than that of the lipid nanoparticles prepared by commercial lipid material SM-102;

[0044] (2) The lipid nanoparticles prepared by the novel ionizable lipid molecule can significantly improve the cell and animal transfection efficiency of the LNP delivery system;

[0045] (3) The disulfide bond and isomannitol structure in the novel ionizable lipid molecule can improve the stability, compatibility, and safety of lipid nanoparticles in vivo;

[0046] (4) The lipid nanoparticles prepared from the novel ionizable lipid molecules provided by the present invention can not only regulate the activity of drugs, increase the drug stability and improve the bioavailability of drugs, but also provide an efficient delivery route for nucleic acid drugs, thus promoting the development of nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 1H NMR spectrum of compound b in Example 1;

[0048] Figure 2 1H NMR spectrum of compound c in Example 1;

[0049] Figure 3 1H NMR spectrum of compound F1 in Example 1;

[0050] Figure 4 1H NMR spectrum of compound F2 in Example 1;

[0051] Figure 5 1H NMR spectrum of compound F3 in Example 1;

[0052] Figure 6 1H NMR spectrum of compound F4 in Example 1;

[0053] Figure 7 1H NMR spectrum of compound F5 in Example 1;

[0054] Figure 8 1H NMR spectrum of compound F6 in Example 1;

[0055] Figure 9 Transfection effect diagrams of F1 to F4 lipid nanoparticles (encapsulating EGFP mRNA) in Example 3 in 293T and A549 cells;

[0056] Figure 10 Transfection effect diagrams of F5, F6 and SM-102 lipid nanoparticles (encapsulating EGFP mRNA) in Example 3 in 293T and A549 cells;

[0057] Figure 11 Transfection effect diagram of lipid nanoparticles at the animal level in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention will be further described in detail below with reference to the drawings and examples. It should be noted that the following examples are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0059] Example 1. Construction of an ionizable lipid molecule containing a disulfide bond and an isomannitol structure

[0060] 1. Synthesis of compound c containing a disulfide bond and an isomannitol structure

[0061] The synthetic route reaction formula is as follows:

[0062]

[0063] 2) Synthesize compound b:

[0064] Dissolve compound a (3 g, 12.6 mmol), which is 4,4'-dithiobutyric acid, in 50 mL of dichloromethane solution, and then add T7 (structural formula as shown in formula T7) (2.1 g, 3.1 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.4 g, 12.6 mmol), and 4-dimethylaminopyridine (770 mg, 6.3 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash with saturated ammonium chloride solution three times and saturated sodium chloride solution once, and dry with anhydrous sodium sulfate. Finally, the product b (2.1 g, 74.3%) is obtained after purification by silica gel column chromatography. 1 1H NMR (400 MHz, Chloroform-d) δ 4.96–4.82 (p, J = 6.2 Hz, 1H), 4.14–3.97 (t, J = 6.6 Hz, 4H), 2.79–2.66 (q, J = 6.7 Hz, 4H), 2.56–2.39 (dt, J = 29.8, 7.3 Hz, 4H), 2.38–2.25 (tt, J = 9.2, 5.3 Hz, 2H), 2.10–1.94 (h, J = 7.4 Hz, 4H), 1.69–1.42 (m, 16H), 1.39–1.14 (m, 48H), 0.92–0.81 (t, J = 6.7 Hz, 12H). The 1H NMR spectrum of compound b is as Figure 1 shown.

[0065] 3) Synthesize compound c:

[0066] Dissolve b (2.1 g, 2.3 mmol) in 30 mL of dichloromethane solution, and then add isomannitol (3.4 g, 23 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.8 g, 9.2 mmol), and 4-dimethylaminopyridine (562 mg, 4.6 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash with saturated ammonium chloride solution three times and saturated sodium chloride solution once, and dry with anhydrous sodium sulfate. Finally, the product c (1.7 g, 71.8%) is obtained after purification by silica gel column chromatography. 1HNMR (400 MHz, Chloroform-d) δ 5.24–5.17 (q, J=5.9 Hz, 1H), 4.96–4.87 (m, 1H), 4.76–4.72 (t, J=5.2 Hz, 1H), 4.50–4.47 (t, J=5.3 Hz, 1H), 4.33–4.27 (t, J=6.9 Hz, 1H), 4.16–3.86 (m, 8H), 3.66–3.62 (s, 2H), 3.60–3.58 (d, J=2.7 Hz, 2H), 2.33–2.27 (m, 2H), 1.77–1.46 (m, 16H), 1.47–1.19 (m, 48H), 0.91–0.85 (t, J=6.7 Hz, 12H). The hydrogen NMR spectrum of Compound c is as Figure 2 shown below.

[0067] 2. Synthesis of Liposome F1

[0068] The reaction scheme of the synthesis route is as follows:

[0069]

[0070] Dissolve Compound c (100 mg, 0.1 mmol) prepared in 1 above in 5 mL of dichloromethane solution, and then add A1 (structural formula as shown in Formula A1) (65.6 mg, 0.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (76.7 mg, 0.4 mmol), and 4-dimethylaminopyridine (24.4 mg, 0.2 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash three times with saturated ammonium chloride solution and once with saturated sodium chloride solution, and dry with anhydrous sodium sulfate. Finally, the product F1 (71.2 mg, 62.3%) is obtained after purification by silica gel column. 11H NMR (400 MHz, Chloroform-d) δ 5.10–4.98 (dtd, J=25.6, 6.6, 5.3 Hz, 2H), 4.87–4.81 (m, 1H), 4.72–4.60 (dt, J=14.5, 5.1 Hz, 2H), 4.07–3.88 (td, J=6.5, 3.5 Hz, 6H), 3.80–3.73 (m, 2H), 3.70–3.51 (m, 10H), 3.11–2.99 (m, 2H), 2.83–2.69 (s, 6H), 2.53–2.48 (t, J=6.6 Hz, 2H), 2.27–2.21 (ddd, J=8.9, 5.2, 3.5 Hz, 2H), 2.17–2.10 (m, 2H), 2.01–1.89 (m, 2H), 1.57–1.36 (m, 16H), 1.24–1.17 (m, 48H), 0.83–0.77 (m, 12H). The hydrogen spectrum of compound F1 is as Figure 3 shown below.

[0071] 3. Synthesis of liposome F2

[0072] The reaction formula of the synthesis route is as follows:

[0073]

[0074] Dissolve the compound c (100 mg, 0.1 mmol) prepared in 1 above in 5 mL of dichloromethane solution, and then add A2 (structural formula shown as formula A2) (92.7 mg, 0.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (76.7 mg, 0.4 mmol), and 4-dimethylaminopyridine (24.4 mg, 0.2 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash it three times with saturated ammonium chloride solution and once with saturated sodium chloride solution, and dry it with anhydrous sodium sulfate. Finally, the product F2 (69.8 mg, 58.3%) is obtained after purification by silica gel column. 1H NMR (400 MHz, Chloroform-d) δ 5.10–5.04 (q, J = 6.1 Hz, 1H), 5.04–4.96 (td, J = 6.8, 5.3 Hz, 1H), 4.88–4.81 (tq, J = 7.2, 2.8 Hz, 1H), 4.72–4.57 (dt, J = 14.6, 5.1 Hz, 2H), 4.02–3.97 (t, J = 6.4 Hz, 4H), 3.67–3.63 (d, J = 7.0 Hz, 4H), 3.61–3.56 (d, J = 11.0 Hz, 2H), 3.54–3.48 (d, J = 11.5 Hz, 2H), 3.23–3.03 (m, 2H), 2.66–2.54 (dq, J = 18.4, 10.1, 9.6 Hz, 2H), 2.43–2.37 (td, J = 7.1, 2.8 Hz, 2H), 2.25–2.22 (m, 2H), 1.95–1.89 (m, 2H), 1.70–1.62 (m, 2H), 1.54–1.47 (m, 8H), 1.27–1.16 (m, 61H), 0.92–0.87 (qt, J = 3.2, 2.1 Hz, 3H), 0.83–0.78 (m, 12H). The 1H NMR spectrum of compound F2 is as shown in Figure 4 shown.

[0075] 4. Synthesis of liposome F3

[0076] The reaction formula of the synthetic route is as follows:

[0077]

[0078] Dissolve the synthesized compound c (100 mg, 0.1 mmol) in 5 mL of dichloromethane solution, and then add A3 (structural formula shown as formula A3) (99.7 mg, 0.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (76.7 mg, 0.4 mmol), and 4-dimethylaminopyridine (24.4 mg, 0.2 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash it three times with saturated ammonium chloride solution and once with saturated sodium chloride solution, and dry it with anhydrous sodium sulfate. Finally, the product F3 (83.1 mg, 68.6%) is obtained after purification by silica gel column. 1H NMR (400 MHz, Chloroform-d) δ 5.10–5.04 (td, J = 6.4, 5.2 Hz, 1H), 5.04–4.96 (td, J = 6.7, 5.3 Hz, 1H), 4.87–4.81 (m, 1H), 4.70–4.60 (dt, J = 15.4, 5.1 Hz, 2H), 4.00–3.96 (d, J = 2.8 Hz, 4H), 3.71–3.62 (q, J = 7.0 Hz, 4H), 3.61–3.55 (d, J = 11.2 Hz, 2H), 3.54–3.49 (d, J = 11.4 Hz, 2H), 3.21–3.08 (s, 2H), 2.81–2.68 (s, 2H), 2.47–2.39 (t, J = 6.8 Hz, 2H), 2.25–2.21 (m, 2H), 2.07–2.01 (s, 2H), 1.54–

[0079] 1.48 (m, 8H), 1.24–1.17 (dt, J = 12.8, 4.0 Hz, 61H), 0.84–0.77 (m, 18H). The 1H NMR spectrum of compound F3 is as shown in Figure 5 Figure.

[0080] 5. Synthesis of liposome F4

[0081] The reaction formula of the synthesis route is as follows:

[0082]

[0083] Dissolve the synthesized compound c (100 mg, 0.1 mmol) in 5 mL of dichloromethane solution, and then add A4 (structural formula shown as formula A4) (93.2 mg, 0.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (76.7 mg, 0.4 mmol), and 4-dimethylaminopyridine (24.4 mg, 0.2 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash it three times with saturated ammonium chloride solution and once with saturated sodium chloride solution, and dry it with anhydrous sodium sulfate. Finally, the product F4 (75.7 mg, 63.2%) is obtained after purification by silica gel column. 1H NMR (400 MHz, Chloroform-d) δ 5.12–5.05 (q, J = 6.1 Hz, 1H), 5.04–4.98 (m, 1H), 4.88–4.82 (td, J = 5.3, 2.6 Hz, 1H), 4.70–4.60 (dt, J = 18.4, 5.1 Hz, 2H), 4.03–3.95 (m, 6H), 3.67–3.61 (q, J = 7.1 Hz, 9H), 3.61–3.57 (d, J = 10.8 Hz, 2H), 3.53–3.49 (d, J = 11.5 Hz, 2H), 2.65–2.56 (s, 4H), 2.40–2.38 (d, J = 4.7 Hz, 4H), 2.37–2.35 (m, 2H), 2.25–2.21 (m, 2H), 1.83–1.75 (p, J = 7.3 Hz, 2H), 1.54–1.48 (m, 8H), 1.20–1.16 (d, J = 6.1 Hz, 54H), 0.83–0.79 (d, J = 6.4 Hz, 12H). The hydrogen spectrum of compound F4 is as shown in Figure 6 shown below.

[0084] 6. Synthesis of liposome F5

[0085] The reaction formula of the synthesis route is as follows:

[0086]

[0087] Dissolve the synthesized compound c (100 mg, 0.1 mmol) in 5 mL of dichloromethane solution, and then add A5 (structural formula shown as formula A5) (79.6 mg, 0.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (76.7 mg, 0.4 mmol), and 4-dimethylaminopyridine (24.4 mg, 0.2 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash it three times with saturated ammonium chloride solution and once with saturated sodium chloride solution, and dry it with anhydrous sodium sulfate. Finally, the product F5 (66.5 mg, 56.8%) is obtained after purification by silica gel column. 1H NMR (400 MHz, Chloroform-d) δ 5.10–5.04 (q, J = 6.2 Hz, 1H), 5.04–4.97 (q, J = 6.3 Hz, 1H), 4.90–4.82 (ddt, J = 9.5, 4.6, 2.2 Hz, 1H), 4.71–4.60 (dt, J = 14.6, 5.0 Hz, 2H), 4.01–3.95 (m, 6H), 3.68–3.61 (q, J = 7.0 Hz, 6H), 3.61–3.58 (d, J = 11.4 Hz, 2H), 3.53–3.49 (d, J = 11.4 Hz, 2H), 3.08–2.99 (q, J = 7.2 Hz, 4H), 2.99–2.93 (m, 2H), 2.51–2.43 (td, J = 6.4, 4.3 Hz, 2H), 2.26–2.20 (td, J = 5.4, 2.6 Hz, 2H), 2.10–2.03 (m, 2H), 1.54–1.48 (m, 8H), 1.22–1.15 (m, 58H), 0.83–0.79 (d, J = 6.2 Hz, 12H). The hydrogen spectrum of compound F5 is as shown in Figure 7 shown.

[0088] 7. Synthesis of Liposome F6

[0089] The reaction formula of the synthesis route is as follows:

[0090]

[0091] Dissolve the synthesized compound c (100 mg, 0.1 mmol) in 5 mL of dichloromethane solution, and then add A6 (structural formula shown as formula A6) (92.6 mg, 0.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (76.7 mg, 0.4 mmol), and 4-dimethylaminopyridine (24.4 mg, 0.2 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash it three times with saturated ammonium chloride solution and once with saturated sodium chloride solution, and dry it with anhydrous sodium sulfate. Finally, the product F6 (72.9 mg, 60.9%) is obtained after purification by silica gel column. 1H NMR (400 MHz, Chloroform-d) δ 5.10–5.04 (q, J = 6.1 Hz, 1H), 5.02–4.95 (m, 1H), 4.88–4.83 (m, 1H), 4.69–4.62 (m, 2H), 4.01–3.96 (td, J = 6.5, 2.1 Hz, 6H), 3.67–3.61 (m, 6H), 3.61–3.56 (d, J = 11.6 Hz, 2H), 3.53–3.49 (d, J = 11.5 Hz, 2H), 3.20–3.10 (d, J = 7.6 Hz, 4H), 3.02–2.96 (d, J = 8.2 Hz, 2H), 2.50–2.44 (t, J = 6.5 Hz, 2H), 2.26–2.21 (ddd, J = 8.9, 5.3, 3.6 Hz, 2H), 2.17–2.09 (m, 2H), 1.94–1.87 (t, J = 5.5 Hz, 4H), 1.68–1.64 (p, J = 2.6 Hz, 4H), 1.55–1.49 (m, 8H), 1.20–1.16 (d, J = 7.3 Hz, 52H), 0.82–0.79 (d, J = 6.4 Hz, 12H). The hydrogen spectrum of compound F6 is as shown in Figure 8 shown.

[0092] 8. Synthesis of liposome F7

[0093] The reaction formula of the synthesis route is as follows:

[0094]

[0095] 1) Synthesis of compound e: Dissolve compound d suberic acid (3 g, 12.6 mmol) in 50 mL of dichloromethane solution, and then add T7 (structural formula shown in formula T7) (2.1 g, 3.1 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.4 g, 12.6 mmol), and 4-dimethylaminopyridine (770 mg, 6.3 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash with saturated ammonium chloride solution three times and saturated sodium chloride solution once, and dry with anhydrous sodium sulfate. Finally, the product e (2.3 g, 72.9%) is obtained after purification by silica gel column chromatography.

[0096] 2) Synthesis of compound c: Dissolve e (2.3 g, 2.3 mmol) in 30 mL of dichloromethane solution, and then add isomannitol (3.4 g, 23 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.8 g, 9.2 mmol), and 4-dimethylaminopyridine (562 mg, 4.6 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash with saturated ammonium chloride solution three times and saturated sodium chloride solution once, and dry with anhydrous sodium sulfate. Finally, the product f (1.6 g, 68.3%) is obtained after purification by silica gel column chromatography.

[0097] 3) Dissolve the compound f (100 mg, 0.1 mmol) prepared in 2) above in 5 mL of dichloromethane solution, and then add A1 (structural formula shown in formula A1) (65.6 mg, 0.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (76.7 mg, 0.4 mmol), and 4-dimethylaminopyridine (24.4 mg, 0.2 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash with saturated ammonium chloride solution three times and saturated sodium chloride solution once, and dry with anhydrous sodium sulfate. Finally, the product F7 (70.2 mg, 61.9%) is obtained after purification by silica gel column chromatography.

[0098] 9. Synthesis of liposome F8

[0099] The reaction formula of the synthesis route is as follows:

[0100]

[0101] Dissolve the compound b (100 mg, 0.1 mmol) prepared in 1 above in 5 mL of dichloromethane solution, and then add A1 (structural formula shown in formula A1) (65.6 mg, 0.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (76.7 mg, 0.4 mmol), and 4-dimethylaminopyridine (24.4 mg, 0.2 mmol) respectively, and react at room temperature for 10 h. After the reaction is completed, wash three times with saturated ammonium chloride solution and once with saturated sodium chloride solution, and dry with anhydrous sodium sulfate. Finally, the product F1 (64.6 mg, 57.7%) is obtained after purification by silica gel column.

[0102] This example only gives the preparation and synthesis process of F1-F8. It can be understood that by changing some of the steps, ionizable lipid molecules containing different T, X, Y, Z, and A groups can be synthesized respectively. For example, by simply replacing the principle T7 used in the synthesis with T6, the ionizable lipid molecules of the T6 group series can be synthesized.

[0103] Example 2: Preparation of liposome nanoparticle complex (LNP preparation)

[0104] The reagents used in the preparation process of the liposome nanoparticle complex are as follows:

[0105] (1) Ionizable lipid molecule: The F1-F8 series of ionizable cationic lipid molecules prepared in Example 1 and the commercial lipid material SM-102 (purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd., China);

[0106] (2) Neutral lipid: DSPC (purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd., China);

[0107] (3) Steroid compound: Cholesterol (purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd., China);

[0108] (4) Polymer-conjugated lipid: DMG-PEG2000 (purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd., China);

[0109] (5) Drug to be delivered: 1) EZ CapTM Firefly Luciferase mRNA (5-OUTP), (purchased from APExBIO, USA), hereinafter referred to as Luc mRNA; 2) EZ CapTM Cy5 EGFP mRNA (5-moUTP), (APExBIO, USA), hereinafter referred to as EGFP mRNA.

[0110] The preparation steps are as follows:

[0111] 1. Dissolve the F1-F8 series of ionizable cationic lipid molecules and SM-102 prepared in Example 1 in absolute ethanol respectively to prepare corresponding solutions with a concentration of 10 mg / mL. Dissolve cholesterol in absolute ethanol to prepare a 5 mg / mL solution, dissolve DSPC in absolute ethanol to prepare a 5 mg / mL solution, and dissolve DMG-PEG2000 in absolute ethanol to prepare a 5 mg / mL solution.

[0112] 2. Mix the F1-F8 series of ionizable cationic lipid molecule solutions and SM-102 solution prepared in Step 1 above with the cholesterol solution, DSPC solution, and DMG-PEG2000 solution respectively according to the mixing ratio of 50:38.5:10:1.5 in molar percentage to form nanoparticles as the alcohol phase, and prepare nine different alcohol phases.

[0113] 3. Add the active ingredients Luc mRNA and EGFP mRNA to 100 mM sodium citrate buffer (pH = 4) respectively, and dilute them to 20 mM with enzyme-free and sterile (DEPC water) as the aqueous phase. Then vortex-mix the nine different alcohol phases prepared in Step 2 with these two aqueous phases according to a volume ratio of 2:1, and dilute them 10-fold with DPBS buffer after stabilizing at room temperature.

[0114] 4. Place the nine alcohol-aqueous mixed phase solutions in Step 3 into 100K ultrafiltration tubes treated with DEPC water respectively, ultrafilter at 3000 rpm for 5 min to remove impurities such as ethanol in the mixed phase solution; dilute the mRNA with DPBS to an appropriate concentration (1 μg / mL).

[0115] 5. After mixing and encapsulating, make the nitrogen-phosphorus ratio of the lipid molecule to mRNA be 6:1, and the preparation of the liposome nanoparticle complex is completed.

[0116] Detect the particle size, polydispersity index, zeta potential and encapsulation efficiency of the nine liposome nanoparticle complexes prepared above. The detection methods are as follows: Use a Malvern Zetasizer Nano ZS ZEN3600 nanoparticle size and zeta potential analyzer (DLS) to detect the particle size, polydispersity index and zeta potential of the lipid nanoparticles at 25 °C, and use an RNA quantification kit Quant-iTTM RiboGreenTM RNA Assay Kit (Thermo Fisher Scientific) to evaluate the encapsulation efficiency of the nanoparticle complex for mRNA.

[0117] The physical and chemical properties such as the particle size, PDI, and zeta potential of the liposome nanoparticle complexes prepared from the F1-F8 series and SM-102 are shown in Table 1 and Table 2 below. Nine liposome nanoparticle complexes in Table 1 encapsulate Luc mRNA, and nine liposome nanoparticle complexes in Table 2 encapsulate EGFP mRNA. The nine liposome nanoparticle complexes encapsulate Luc mRNA or EGFP mRNA mixed in the same mass ratio.

[0118] Table 1. Particle size, polydispersity index, zeta potential, and encapsulation efficiency of Luc mRNA of different lipid nanoparticles

[0119]

[0120] Table 2. Particle size, polydispersity index, zeta potential, and encapsulation efficiency of EGFP mRNA of different lipid nanoparticles

[0121]

[0122] According to the data analysis in Table 1 and Table 2, the particle size, particle size distribution, and zeta potential of the lipid nanoparticles prepared from F1-F6 provided by the present invention are comparable to those of the lipid nanoparticles prepared from the commercial lipid material SM-102, fully meeting the basic conditions for mRNA delivery. Moreover, due to their good water solubility, the lipid nanoparticles prepared from F1-F6 have a higher encapsulation efficiency for both Luc mRNA and EGFP mRNA than the lipid nanoparticles prepared from the commercial lipid material SM-102.

[0123] Comparing the encapsulation efficiencies of the lipid nanoparticles containing F1, F7, and F8 for Luc mRNA and EGFP mRNA, it shows that the encapsulation efficiency of the lipid nanoparticles containing F1 is significantly higher than that of the lipid nanoparticles containing F7 and F8. Compared with F1, F7 has one less disulfide bond structure in its ionizable lipid molecule. The disulfide bond can form a cross-linked structure in the lipid nanoparticles, making the lipid nanoparticles more stable; compared with F1, F8 has one less isomannitol structure in its ionizable lipid molecule, which has the potential to interact with the phospholipid molecules of the liposome. It is speculated that it may not only enhance the stability of the liposome bilayer membrane through interactions such as hydrogen bonds, but also may change the fluidity of the liposome bilayer membrane. A liposome with good fluidity can more flexibly adjust its structure to adapt to drug molecules of different sizes and shapes, thereby more tightly encapsulating the drug and reducing the possibility of drug leakage from the liposome. The combined addition of the disulfide bond structure and the isomannitol structure improves the stability and fluidity of the lipid nanoparticle bilayer membrane, and thus improves the encapsulation efficiency of mRNA.

[0124] Therefore, the simultaneous presence of the disulfide bond and the isomannitol structure in the F1-F6 ionizable lipid molecules can significantly improve the encapsulation efficiency of the lipid nanoparticles prepared therefrom.

[0125] Example 3: Transfection Experiments at the Cellular and Animal Levels

[0126] 1. Conduct transfection experiments at the cellular level using the lipid nanoparticles prepared in Example 2

[0127] The cell lines used in the experiment were: 1) human non-small cell lung cancer cell line (A549 cell line), cultured in RPMI 1640 complete medium supplemented with 10% fetal bovine serum and 1% antibiotics; 2) human embryonic kidney cell line (293T cell line), cultured in DMEM complete medium supplemented with 10% fetal bovine serum and 1% antibiotics.

[0128] The experiment was divided into two groups:

[0129] The first group: Add the nine lipid nanoparticles (encapsulating Luc mRNA) in Table 1 to the cultured 293T and A549 cells respectively;

[0130] The second group: Add the nine lipid nanoparticles (encapsulating EGFP mRNA) in Table 2 to the cultured 293T and A549 cells respectively.

[0131] The experimental procedure was as follows: Use a cell counter to adjust the 293T and A549 cells cultured in the medium to 2.7×10 6 cells / mL, inoculate them in a 96-well plate, add 100 μL of the lipid nanoparticle solution to each well and culture overnight. After the cells adhered and grew to 80% of the well plate area, discard the original medium. Detect the fluorescence intensity of EGFP mRNA in the second group under the excitation of 450 nm blue light using an OLYMPUS CKX53 microscope, and detect Luc mRNA in the first group using a Tecan multifunctional microplate reader.

[0132] The results of detecting the fluorescence intensity of EGFP mRNA in the second group are as Figure 9 - 10 shown. The results show that in both 293T cells and A549 cells, the lipid nanoparticles containing F1, F4, and F5 have better cell transfection effects than the other four. After transfection into cells, they make the cells brighter, indicating that the higher the EGFP content in the cells, and also indicating that the lipid nanoparticles containing F1, F4, and F5 can effectively deliver mRNA.

[0133] The results of detecting Luc mRNA in the first group are shown in Table 3-4 below. Table 3 shows the absorbance values of the cells after the nine lipid nanoparticles in Table 1 were transfected into 293T cells, and Table 4 shows the absorbance values of the cells after the nine lipid nanoparticles in Table 1 were transfected into A549 cells. The higher the absorbance value, the better the transfection effect of the lipid nanoparticles.

[0134] Table 3. Absorbance values of different lipid nanoparticles encapsulating Luc mRNA transfected into 293T cells

[0135] Name Absorbance F1 47236 F2 13547 F3 6845 F4 43548 F5 39784 F6 8648 F7 17863 F8 15472 SM-102 39551

[0136] Table 4. Absorbance values of different lipid nanoparticles encapsulating Luc mRNA transfected into A549 cells

[0137] Name Absorbance F1 98465 F2 32545 F3 24532 F4 94530 F5 87865 F6 34535 F7 24792 F8 17238 SM-102 78460

[0138] According to the data analysis in Tables 3 - 4, by comparing the absorbance values of lipid nanoparticles containing F1 - F6 after transfection into 293T cells or A549 cells, the lipid nanoparticles containing F1, F4, and F5 have better transfection effects than the other lipid nanoparticles. The difference among F1 - F6 lies in that the hydrophilic part of the ionizable lipid molecule contains different structures A, indicating that the three structures A of F1 (its structure A is shown as formula A1), F4 (its structure A is shown as formula A4), and F5 (its structure A is shown as formula A5) can improve the cell transfection rate of lipid nanoparticles more than the three structures A of F2 (its structure A is shown as formula A2), F3 (its structure A is shown as formula A3), and F6 (its structure A is shown as formula A6).

[0139] The absorbance values of lipid nanoparticles containing F1, F4, and F5 after transfection into 293T cells or A549 cells are significantly higher than those of lipid nanoparticles containing SM - 102, and the absorbance value of lipid nanoparticles containing F1 is the highest, indicating that lipid nanoparticles containing F1, F4, and F5 have better transfection efficiency than lipid nanoparticles containing SM - 102, and lipid nanoparticles containing F1 have the highest transfection efficiency.

[0140] Comparing the transfection effects of lipid nanoparticles containing F1, F7, and F8 in the above two tables, the transfection efficiency of lipid nanoparticles containing F1 is significantly higher than that of lipid nanoparticles containing F7 and F8. Compared with F1, F7 lacks a disulfide bond structure in its ionizable lipid molecule, and F8 lacks an isomannitol structure in its ionizable lipid molecule compared with F1. Introducing a disulfide bond into the liposome structure can not only enhance the overall stability of the liposome but also enable the liposome to release the loaded drug under specific reducing conditions to achieve precise drug delivery; the addition of isomannitol may regulate its permeability by changing the fluidity or permeability of the liposome bilayer membrane, and this regulatory effect helps to achieve controlled and sustained release of the drug and improve the drug delivery efficiency of the liposome. Combining the disulfide bond structure and the isomannitol structure can significantly improve its transfection efficiency and bioavailability by enhancing the stability, permeability, and biocompatibility of lipid nanoparticles.

[0141] 2. Perform in vivo transfection experiments on the lipid nanoparticles containing F1, F4, F5, and SM-102 with high cell transfection efficiency

[0142] The mice used in the experiment were 6-8-week-old C57BL / 6 mice from Hangzhou Hangsheng Biotechnology Co., Ltd.

[0143] The specific experimental steps were as follows: The above four lipid nanoparticles encapsulating Luc mRNA were respectively injected intramuscularly or intravenously into four groups of mice at a dose of 2 μg / mouse, with 3 mice in each group. At 6 h after injection, each group of mice was respectively injected with 200 μL of D-luciferin sodium solution (1.5 mg / mL -1 ), and then anesthetized with isoflurane in an anesthesia machine. After anesthesia, the mice were placed in a small animal imaging system (PerkinElmer IVIS Lumina III) to detect bioluminescence, and the detection time was for automatic exposure.

[0144] The detection results were as Figure 11 shown. The results showed that all of the above four lipid nanoparticles encapsulating Luc mRNA had transfection effects. After transfection of mice with the lipid nanoparticles containing F1 and SM-102, the imaging showed that the transfected area was larger and the transfection concentration was higher than those of the other two. When comparing the transfection effects of the two lipid nanoparticles containing F1 and SM-102 in mice, the transfection effect of the lipid nanoparticles containing F1 in mice was better than that of the lipid nanoparticles containing SM-102, which was the best.

[0145] In summary, the lipid nanoparticles containing F1 have extremely high transfection efficiencies at the cell level and in vivo, and can effectively deliver mRNA into cells or organisms. Preferably, F1 is used as the ionizable lipid molecule for preparing lipid nanoparticles.

[0146] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be determined by the scope defined by the claims.

Claims

1. An ionizable lipid molecule, characterized in that has a structure as shown in formula I, or contains a structure as shown in formula I or a derivative thereof, Wherein, T is selected from C 3-20 Alkyl single chain, or double chain with carbon number greater than or equal to 3, one of the double chains is selected from -(CH2) a C=C(CH2) b C=C(CH2) c , or -(CH2) a OC(=O)(CH2) b ,or a, b, c are independently selected from integers from 1 to 10; X is selected from -CH2OC(=O)NH-, -CH2OC(=O)O-, -CH2C(=O)O-, -CH2C(=O)NH-, Any one of -CH2C(=O)S-, -CH2O-; Y is selected from any substituted or unsubstituted C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C 3-8 Cycloalkenyl, C 3-8 Cycloalkynyl, 4-8 membered heterocyclic ring; Z is selected from -O(C=O)(CH2) n -、-O(CH2) n -, -O(C=O)O-, -O(C=O)-, -O(C=O)O(CH2) n -middle Any one of , wherein n is an integer between 1 and 7; A is selected from R1 and R2 are each independently selected from any substituted or unsubstituted C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C 3-8 Cycloalkenyl, C 3-8 Cycloalkynyl, phenyl, -(C=O)C 1-3 Alkyl; or R1 and R2 combine to form an optionally substituted or unsubstituted 4-8 membered heterocyclic ring, pyrimidine ring, purine ring; wherein the substituent group is 1 or 2 or 3 or 4 or 5 independent OH, SH, nitro, cyano, amino, C 1-3 Hydroxyl, C 1-3 Alkoxy, -(C=O)OC 1-3 Alkyl, C 1-3 Alkyl; M is selected from N atoms or C atoms.

2. The ionizable lipid molecule according to claim 1, wherein The structural formula of Y is:

3. The ionizable lipid molecule according to claim 2, wherein The T is selected from any one of the structures shown in formulas T1-T7:

4. The ionizable lipid molecule according to claim 3, wherein The A is selected from any one of the structures shown in formula A1-A18:

5. The ionizable lipid molecule according to claim 4, wherein The structural formula of T is shown in formula T7; the structural formula of A is selected from any one or more of the structures shown in formulas A1, A2, A3, A4, A5, and A6.

6. The ionizable lipid molecule according to claim 5, characterized in that The structural formula of X is: -CH2C(=O)O-; the structural formula of Z is: -O(C=O)(CH2)3-.

7. The ionizable lipid molecule according to claim 6, wherein Including any one or more of formulas F1-F6:

8. A nanoparticle composition, characterized in that: The invention comprises an ionizable lipid molecule, a neutral lipid, a steroid compound, or a polymer conjugate as described in any one of claims 1 to 7.

9. Use of the nanoparticle composition as claimed in claim 8 for preparing a delivery vehicle.

10. Use of the ionizable lipid molecule according to any one of claims 1 to 7 for preparing a preparation for improving drug transfection efficiency, characterized in that: The medicament includes a therapeutic or prophylactic agent.

Images