WO2022170835A1 - 可电离的阳离子脂质类似物材料在作为核酸药物递送载体或转染试剂中的应用 - Google Patents

可电离的阳离子脂质类似物材料在作为核酸药物递送载体或转染试剂中的应用 Download PDF

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WO2022170835A1
WO2022170835A1 PCT/CN2021/136192 CN2021136192W WO2022170835A1 WO 2022170835 A1 WO2022170835 A1 WO 2022170835A1 CN 2021136192 W CN2021136192 W CN 2021136192W WO 2022170835 A1 WO2022170835 A1 WO 2022170835A1
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cationic lipid
i2r3c18
alkyl group
nucleic acid
lipid analog
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刘志佳
乐志成
陈永明
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中山大学
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    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
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Definitions

  • the present invention relates to the technical field of biomedicine, in particular to the application of an ionizable cationic lipid analog material as a nucleic acid drug delivery carrier or a transfection reagent.
  • Nucleic acid drugs refer to DNA or RNA with disease treatment functions. They have the advantages of strong design, good specificity, and resistance to drug resistance. They are currently widely used in protein replacement therapy, gene editing, nucleic acid vaccines, etc. Nucleic acid drugs are unstable and are rapidly degraded by nucleases in the blood or cleared by the kidneys. In addition, the non-specific distribution of nucleic acid drugs will reduce the local concentration of the target tissue, and the nucleic acid entering the cell must successfully avoid and/or escape the endosome and enter the cytoplasm before it can function. Therefore, nucleic acid drugs need to be delivered by various types of carriers. Viral vectors have the advantages of short action time and high transfection efficiency. They are relatively mature nucleic acid drug delivery vectors.
  • viruses used for transfection have been specially treated, their genomes will theoretically not be copied and inserted into the host genome. , but the virus is prone to gene mutation, and the treated virus may still recover to the wild-type ability to copy its own genes. In addition, the virus is immunogenic and can easily lead to an immune response. Therefore, viral vectors have certain safety hazards in the process of preparation and use, which greatly limits their application. It is necessary to further develop non-viral vectors for efficient and safe delivery of nucleic acid drugs.
  • the purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of ionizable cationic lipid analog materials as nucleic acid drug delivery carriers or transfection reagents.
  • the cationic lipid analog material has a structure as shown in formula (I):
  • m 1 is independently selected from branched alkyl, phenyl or heteroatom-containing aryl;
  • R 1 is an alkyl group
  • R 2 is an alkyl group
  • R 3 is an alkyl group or a phenyl group, or R 2 and R 3 are connected to a cyclic group or a heterocyclic group
  • n is independently selected from linear alkyl, linear alkenyl or
  • m 4 is independently selected from a linear alkyl group, an ether bond-containing linear alkyl group, or an N-containing heterocyclic ring-containing alkyl group.
  • the cationic lipid analog material of the present invention can be complexed with it to form a stable complex to achieve efficient intracellular delivery, and the nucleic acid delivered into the cell can be released from the complex to achieve Expression of the transgene or silencing of the target gene.
  • the cationic lipid analog material designed in the present invention can be used as a nucleic acid drug delivery carrier or a transfection reagent, and a universal, efficient and low-toxic nucleic acid drug delivery system can be developed, and has practical biomedical application value.
  • delivery or "intracellular delivery” as used herein refers to the entry of nucleic acid from the outside of the cell into the interior of the cell, so that it is confined in the cytosol or in the organelle of the cell.
  • m 1 is an alkyl, phenyl or heteroatom-containing aryl group substituted by a substituent ⁇ , and the substituent includes a methyl group, and further, m 1 is
  • m 3 is a straight-chain alkyl group with 7-19 carbon atoms, a straight-chain alkenyl group with 17 carbon atoms or Further, m3 is
  • m 4 is a straight-chain alkyl group with 6 carbon atoms, a straight-chain alkyl group with 4-8 carbon atoms containing an ether bond, or an alkyl group containing an N-containing heterocycle.
  • the described cationic lipid analog material has any one of the following 72 structures:
  • the delivery effect of nucleic acid is related to the structure of the cationic lipid analog material, and it is found that the above-mentioned 72 small molecule cationic lipid analog materials can be combined with various nucleic acid molecules to form nanoparticles , to achieve efficient intracellular delivery of plasmid DNA, mRNA and siRNA, and the above-mentioned cationic lipid analog materials have low cytotoxicity.
  • the present invention adopts the plasmid DNA expressing green fluorescent protein (GFP) or the plasmid DNA expressing luciferase (luminescence) as the reporter gene, and detects the gene transfection efficiency of different cationic lipid analog materials in HeLa cells.
  • GFP green fluorescent protein
  • luciferase luminescence
  • the cationic lipid analog material is at least one of I2R2C18-1A1, I2R2C18-2A1, I2R3C18-1A1, and I2R3C18-2A1.
  • the present invention detects the siRNA transfection and delivery effects of cationic lipid analog materials in A549 cells (A549-Luc), and the above four materials can deliver siRNA, which can achieve efficient gene silencing.
  • the nucleic acid used in the present invention is not particularly limited to its type or structure, and the nucleic acid includes but is not limited to messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA ( At least one of miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), plasmid DNA (pDNA), small circular DNA, and genomic DNA (gNDA).
  • the nucleic acid used in the present invention may be a nucleic acid derived from humans, animals, plants, bacteria, viruses, etc., or a nucleic acid prepared by chemical synthesis.
  • nucleic acid may be any of single-stranded, double-stranded and triple-stranded, and its molecular weight is not particularly limited.
  • nucleic acids of the present invention may be chemically, enzymatically or peptide-modified nucleic acids.
  • the present invention also provides a method for preparing the above cationic lipid analog material.
  • the specific method is as follows: adding an aldehyde compound and an amine compound into an organic solution, and then adding a carboxylic acid compound and an isocyanide compound in sequence after the reaction for 10-120 minutes, The reaction is carried out at 4-60° C. for 6-72 hours. After the reaction, the product is separated and purified by a chromatographic column to obtain the cationic lipid analog material.
  • the invention adopts aldehyde compounds, amine compounds, carboxylic acid compounds and isocyanide compounds as raw materials, and synthesizes small molecule cationic lipid analog materials through Ugi reaction.
  • the cationic lipid analog material of the present invention has mild reaction conditions, simple synthesis process, good stability, no complicated reaction device, low toxicity of the synthesized small molecule cationic lipid analog material, high transfection efficiency, and can convert nucleic acid into Drug delivery into cells with high efficiency.
  • a chromatographic column separation is performed under the condition that a mixture of methanol and dichloromethane is used as a mobile phase.
  • the molar ratio of the aldehyde compound, the amine compound, the carboxylic acid compound and the isocyanide compound is 0.1-1:0.1-1:0.1-1 : 0.1-1; further, the molar ratio is 1:1:1:0.5.
  • the aldehyde compound is selected from any one of the following compounds A1-A3:
  • aldehyde compound is preferably an A1 compound
  • the amine compound is selected from any one of the following compounds R1-R11:
  • the carboxylic acid compound is selected from any one of the following compounds CHS, C18-1, C18-2 or C8-C20:
  • the isocyanide compound is selected from any one of the following compounds I1, I2-1, I2, I2-3, and I3:
  • the cationic lipid analog material of the present invention can efficiently bind plasmid DNA, mRNA, siRNA, etc., and it can deliver different plasmid DNAs on a variety of cells, all of which have high transfection efficiency, even reaching or higher than the current commercial level of transfection reagent.
  • the cationic lipid analog material of the present invention can be used as a safe and efficient nucleic acid drug intracellular delivery carrier or transfection reagent, and has practical biomedical application value.
  • Fig. 1 is the mass spectrum (a) and proton NMR spectrum (b) of the cationic lipid analog material I2-1R2C18A1.
  • FIG. 2 is the mass spectrum (a) and proton NMR spectrum (b) of the cationic lipid analog material I2R2C18A1.
  • FIG. 3 is the mass spectrum (a) and proton NMR spectrum (b) of the cationic lipid analog material I2-3R2C18A1.
  • Figure 4 shows the results of the positive rate of plasmid DNA expressing GFP transfected with different cationic lipid analog materials.
  • Figure 5 shows the detection results of luciferase expression in HeLa cells after transfection of luciferase-expressing plasmid DNA with different cationic lipid analog materials.
  • Figure 6 shows the results of laser confocal transfection of GFP-expressing plasmid DNA into different types of cells with different added amounts of I2R3C18-1A1.
  • Figure 7 shows the results of the positive rate of eGFP-mRNA transfected with different cationic lipid analog materials.
  • Figure 8 shows the mean fluorescence intensity of eGFP-mRNA transfected with different cationic lipid analog materials. Among them, the experimental conditions are consistent with Fig. 7.
  • Figure 9 shows the laser confocal results of I2R2C18-2A1, I2R3C18-2A1, and I2R11C18-2A1 transfecting eGFP-mRNA into DC2.4 cells.
  • the commercial transfection reagent Lipofectamine 2000 was used as a positive control.
  • Figure 10 shows the gene silencing results of siRNA transfected with different amounts of I2R2C18-1A1, I2R3C18-1A1, I2R2C18-2A1, and I2R3C18-2A1.
  • Figure 11 shows the cytotoxicity results of I2R2C16A1, I2R2C17A1, I2R2C18A1, I2R2C19A1, and I2R2C20A1.
  • the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.
  • the synthetic route of the cationic lipid analog material of the present invention is:
  • the amine compound m 2 -NH 2 is any one of the following compounds R1-R11; carboxylic acid compound It is any one of the following compounds C8-C20 and CHS; aldehyde compounds is any one of the following compounds A1-A3; isocyanide compounds is any one of the following compounds I1-I3;
  • the specific preparation method of the cationic lipid analog material of the present embodiment is as follows: 1 mmol of isobutyraldehyde and 1 mmol of amine compound are respectively added to 0.5 mL of methanol solution, and 1 mmol of carboxylic acid compound and 0.5 mmol of carboxylic acid are added in sequence after the reaction for 60 min. The isocyanide compound was reacted at 40° C. for 12 hours. After the reaction, the product was separated and purified by a chromatographic column, wherein the mobile phase was a mixture of methanol and dichloromethane.
  • Cationic lipid analogs I2-1R2C18A1, I2R2C18A1 and I2-3R2C18A1 were selected as expression materials, and their structures were characterized. Among them, the mass spectrum and H NMR spectrum of I2-1R2C18A1 are shown in Figure 1; the mass spectrum and H NMR spectrum of I2R2C18A1 are shown in Figure 2; the mass spectrum and H NMR spectrum of I2-3R2C18A1 The spectrum The diagram is shown in Figure 3. The results of 1H NMR and MS were consistent with the expected structure of the cationic lipid analog material.
  • plasmid DNA expressing green fluorescent protein was used as a reporter gene to detect the gene transfection efficiency of cationic lipid analog materials in HeLa cells.
  • GFP green fluorescent protein
  • HeLa cells were seeded on 24-well plates and cultured in a cell incubator for 12 h.
  • Different cationic lipid analog materials 0.25-8 ⁇ g/well
  • GFP-expressing plasmid DNA 0.5 ⁇ g/well
  • were incubated at 40 After mixing in microliter sodium acetate buffer (25mM, pH 5.2), let stand for 10 minutes, and then dilute into 460 microliters Opti-MEM medium to obtain cationic lipid analog complex particle solution loaded with plasmid DNA.
  • the medium of HeLa cells was removed, washed with PBS and then added with the compounded particle solution. After culturing for 24 hours, the transfection efficiency of plasmid DNA in the cells was analyzed by flow cytometry.
  • the commercial gene transfection reagent Lipofectamine 2000 was used as a positive control.
  • the cationic lipid analog material of the present invention can transfect GFP-expressing plasmid DNA into HeLa cells, especially I1R2C14A1, I1R2C18-2A1, I1R11C14A1, I2R1C14A1, I2R1C16A1, I2R1C18-1A1, I2R1C18-2A1, I2R2C14A1 ⁇ I2R2C16A1 ⁇ I2R2C18-1A1 ⁇ I2R2C18-2A1 ⁇ I2R3C16A1 ⁇ I2R3C18A1 ⁇ I2R3C18-1A1 ⁇ I2R3C18-2A1 ⁇ I2R11C14A1 ⁇ I2R11C16A1 ⁇ I2R11C18A1 ⁇ I2R11C18-1A1 ⁇ I2R11C18-1A1 ⁇ I2R11C18-2A1 ⁇ Lipofectamine 2000.
  • the reporter gene expressing luciferase was used as the model plasmid DNA, and the gene transfection efficiency of the cationic lipid analog material was detected in HeLa cells.
  • the specific operation method was as follows: HeLa cells were seeded on 96-well plates and cultured Incubate for 12 hours, mix different cationic lipid analog materials and luciferase-expressing plasmid DNA (0.5 ⁇ g) in 40 ⁇ l sodium acetate buffer (25 mM, pH 5.2) and let stand for 10 minutes, respectively. Dilute into 460 microliters of Opti-MEM medium to obtain the cationic lipid analog complex particle solution loaded with plasmid DNA.
  • the medium of HeLa cells was removed, and 125 ⁇ l of the compounded particle solution was added after washing with PBS. After culturing for 24 hours, the culture solution was discarded, 50 ⁇ l/well of substrate was added after fully lysing, and the expression level of luciferase was detected using a multifunctional microplate reader.
  • Example 4 The effect of I2R3C18-1A1 transfection of GFP-expressing plasmid DNA into different types of cells
  • I2R3C18-1A1 was used as a representative cationic lipid analog material
  • GFP-expressing plasmid DNA was used as a reporter gene to explore the effect of different dosages of cationic lipid analog materials (0.5-3 ⁇ g/well) in different types of cells. Plasmid DNA (0.5 ⁇ g/well) within the transfection effect.
  • the commercial gene transfection reagent Lipofectamine 2000 was used as a positive control.
  • the I2R3C18-1A1 particle solution loaded with plasmid DNA expressing GFP was prepared by referring to the method in Example 8, and the solution was prepared in mouse dendritic cells (DC 2.4), mouse macrophages (RAW264.7), adenocarcinoma human alveoli, respectively.
  • Basal epithelial cells (A549), human pancreatic cancer cells (BxPC3) and HeLa cells were added with I2R3C18-1A1 particle solution complex solution loaded with plasmid DNA expressing GFP, and after culturing for 24 hours, the intracellular plasmid was observed by laser confocal microscope DNA transfection efficiency.
  • Figure 6 shows that the I2R3C18-1A1 material can transfect GFP-expressing plasmid DNA to tumor cells and immune cells.
  • eGFP-mRNA was selected as the model mRNA, and the mRNA transfection efficiency of the cationic lipid analog material was detected in DC 2.4 cells.
  • the specific operation method is as follows:
  • DC 2.4 cells were seeded on 48-well plates and cultured in a cell incubator for 12 h.
  • Different cationic lipid analog materials (0.25–2 ⁇ g/well) and eGFP-expressing mRNA (0.2 ⁇ g/well) were incubated at 20 Mix in microliter sodium acetate buffer (25mM, pH 5.2), let stand for 10 minutes, and then dilute into 230 microliter Opti-MEM medium to obtain mRNA-loaded composite particles.
  • the medium of HeLa cells was removed, washed with PBS and then added with the composite particle solution. After culturing for 24 hours, the mRNA transfection efficiency in the cells was observed by flow cytometry and laser confocal microscopy.
  • the commercial gene transfection reagent Lipofectamine 2000 was used as a positive control.
  • Figures 7-9 show that the cationic lipid analog materials of the present invention can transfect eGFP-expressing mRNA into DC 2.4 cells, especially I1R2C14A1, I1R2C16A1, I1R2C18A1, I1R2C18-1A1, I1R2C18-2A1, I1R11C14A1, I1R11C16A1, I1R11C18A1, I2R1C14A1 ⁇ I2R1C16A1 ⁇ I2R1C18-1A1 ⁇ I2R2C14A1 ⁇ I2R2C16A1 ⁇ I2R2C18-1A1 ⁇ I2R2C14A1 ⁇ I2R2C16A1 ⁇ I2R2C18-1A1 ⁇ I2R2C18-2A1 ⁇ I2R3C14A1 ⁇ I2R3C16A1 ⁇ I2R3C18A1 ⁇ I2R3C18-1A1 ⁇ I2R3C18-2A1 ⁇ I2R3C14A1 ⁇ I
  • I2R2C18-1A1, I2R3C18-1A1, I2R2C18-2A1, and I2R3C18-2A1 were selected as representative cationic lipid analog materials, and the siRNA transfection and The delivery effect is as follows:
  • A549 cells expressing luciferase (A549-Luc) were seeded in 96-well plates and incubated in a cell incubator for 12 hours, and the different materials (0.5–3 ⁇ g/well) and siRNA were mixed in 40 ⁇ l of sodium acetate buffer.
  • I2R2C16A1, I2R2C18A1, I2R2C17A1, I2R2C19A1, I2R2C20A1 with higher transfection efficiency were selected as representative cationic lipid analog materials, and the toxicity of cationic lipid analog materials to HeLa cells was detected by MTT assay.
  • the specific experimental method is as follows: HeLa cells were plated on 96-well plates and incubated in a cell incubator for 12 hours, then the cell culture medium was removed and replaced with 1 ⁇ g/ml cationic lipidoid material and incubated for 4 hours, then the material was washed off and Changed to DMEM medium and cultured for 20 hours, and finally detected the cell viability using MTT.
  • Figure 11 shows that the cationic lipid analog materials I2R2C16A1, I2R2C17A1, I2R2C18A1, I2R2C19A1, and I2R2C20A1 of the present invention are less cytotoxic and have good biocompatibility.
  • the prepared cationic lipid analog material also has the characteristics of low cytotoxicity, using plasmid DNA expressing green fluorescent protein (GFP) or plasmid DNA expressing luciferase (luminescence) as reporter gene, and detected in HeLa cells Gene transfection efficiency of cationic lipid analog materials, which also have some effect. Therefore, it can be speculated that these cationic lipid analog materials can also be used as nucleic acid drug delivery vehicles.
  • GFP green fluorescent protein
  • luciferase lumine

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Abstract

本发明公开了可电离的阳离子脂质类似物材料在作为核酸药物递送载体或转染试剂中的应用。本发明的阳离子脂质类似物材料可以高效结合并递送质粒DNA、mRNA、siRNA等核酸分子，实现高效的基因转染或基因沉默，且细胞毒性低。本发明的阳离子脂质类似物材料可作为安全、高效的核酸药物胞内递送载体或转染试剂，具有实际的生物医学应用价值。

Description

可电离的阳离子脂质类似物材料在作为核酸药物递送载体或转染试剂中的应用 技术领域
本发明涉及生物医药技术领域，尤其是涉及可电离的阳离子脂质类似物材料在作为核酸药物递送载体或转染试剂中的应用。
背景技术
核酸药物是指具有疾病治疗功能的DNA或RNA，具有设计性强、特异性好、不易产生耐药性等优点，目前广泛应用于蛋白替代疗法、基因编辑、核酸疫苗等。核酸药物不稳定，会快速被血液中的核酸酶降解或被肾脏清除。并且，核酸药物的非特异性分布会降低靶组织的局部浓度，进入细胞的核酸还要成功避开和/或逃逸内涵体进入细胞质，才能发挥作用。因此，核酸药物需要借助各类载体实现药物递送。病毒类载体具有作用时间短、转染效率高等优点，是目前研究较为成熟的核酸药物递送载体，用于转染的病毒虽已经过特殊处理，其基因组理论上不会拷贝和插入到宿主基因组中，但是病毒容易发生基因突变，被处理过的病毒仍有可能恢复到野生型拥有拷贝自身基因的能力。此外，病毒具有免疫原性，容易导致免疫反应。因此，病毒类载体在制备和使用过程中存在一定的安全隐患，很大限制了其应用，需要进一步开发高效和安全输送核酸药物的非病毒载体。
发明内容
本发明的目的在于克服现有技术的不足之处而提供可电离的阳离子脂质类似物材料在作为核酸药物递送载体或转染试剂中的应用。
为实现上述目的，本发明采取的技术方案如下：
可电离的阳离子脂质类似物材料在作为核酸药物递送载体或转染试剂中的应用，所述阳离子脂质类似物材料具有如式(I)所示结构：
Figure PCTCN2021136192-appb-000001
式(I)中，m 1独立地选自支链烷基、苯基或含杂原子的芳香基；
m 2为
Figure PCTCN2021136192-appb-000002
R 1为烷基，R 2为烷基，R 3为烷基或苯基，或R 2与R 3连接为环基或杂环基；
m 3独立地选自直链烷基、直链烯基或
Figure PCTCN2021136192-appb-000003
m 4独立地选自直链烷基、含醚键的直链烷基或含有含N杂环的烷基。
无论是质粒DNA、mRNA还是siRNA，本发明的阳离子脂质类似物材料均能与其复合形成稳定的复合物，实现高效的胞内递送，并且递送至细胞内的核酸能够从复合物释放出来，实现转入基因的表达或对目标基因的沉默。本发明设计的阳离子脂质类似物材料可作为核酸药物递送载体或转染试剂，可以开发出普适、高效、低毒的核酸药物递送系统，具有实际的生物医学应用价值。
需要说明的是，本文所用术语“递送”或者“胞内递送”，指的是使核酸从细胞的外部进入到细胞的内部，使其局限在细胞溶质中或在细胞的细胞器内。
进一步地，上述式(I)中，m 1为被取代基α取代的烷基、苯基或含杂原子的芳香基，所述取代基包括甲基，更进一步地，m 1为
Figure PCTCN2021136192-appb-000004
进一步地，上述式(I)中，m 2为
Figure PCTCN2021136192-appb-000005
Figure PCTCN2021136192-appb-000006
获得的材料转染效率较高。
进一步地，上述式(I)中，m 3为碳原子数为7-19的直链烷基、碳原子数为17的直链烯基或
Figure PCTCN2021136192-appb-000007
进一步地，m 3为
Figure PCTCN2021136192-appb-000008
Figure PCTCN2021136192-appb-000009
Figure PCTCN2021136192-appb-000010
进一步地，上述式(I)中，m 4为碳原子数为6的直链烷基、碳原子数为4-8的含醚键的直链烷基或含有含N杂环的烷基。
进一步地，所述的阳离子脂质类似物材料具有如下72种结构中的任一种：
Figure PCTCN2021136192-appb-000011
Figure PCTCN2021136192-appb-000012
本发明通过不同阳离子脂质类似物材料的筛选，核酸的递送效果与阳离子脂质类似物材料的结构有关，发现上述72种小分子阳离子脂质类似物材料可以与多种核酸分子结合形成纳米粒，实现高效的质粒DNA、mRNA和siRNA的胞内递送，且上述阳离子脂质类似物材料的细胞毒性较低。
进一步地，所述阳离子脂质类似物材料为I1R2C14A1、I1R2C18-2A1、I1R11C14A1、I2R1C14A1、I2R1C16A1、I2R1C18-1A1、I2R1C18-2A1、I2R2C14A1、I2R2C16A1、I2R2C18-1A1、I2R2C18-2A1、I2R3C16A1、I2R3C18A1、I2R3C18-1A1、I2R3C18-2A1、I2R11C14A1、I2R11C16A1、I2R11C18A1、I2R11C18-1A1、I2R11C18-2A1中的至少一种。本发明采用表达绿色荧光蛋白(GFP)的质粒DNA或者表达荧光素酶(luminescence)的质粒DNA作为报告基因，在HeLa细胞检测不同阳离子脂质类似物材料的基因转染效率，结果表明，上述20种阳离子脂质类似物材料具有较高的转染效率，其转染效率达到或高于商品化转染试剂Lipofectamine2000。
进一步地，所述阳离子脂质类似物材料为I1R2C14A1、I1R2C16A1、I1R2C18A1、I1R2C18-1A1、I1R2C18-2A1、I1R11C14A1、I1R11C16A1、I1R11C18A1、I2R1C14A1、I2R1C16A1、I2R1C18-1A1、I2R2C14A1、I2R2C16A1、I2R2C18A1、I2R2C18-1A1、I2R2C18-2A1、I2R3C14A1、I2R3C16A1、I2R3C18A1、I2R3C18-1A1、I2R3C18-2A1、I2R11C14A1、I2R11C16A1、I2R11C18A1、I2R11C18-1A1、I2R11C18-2A1中的至少一种。本发明选用eGFP-mRNA作为模型mRNA，在DC 2.4细胞比较不同阳离子脂质类似物材 料的mRNA转染效率，上述阳离子脂质类似物材料具有较高的转染效率。
进一步地，所述阳离子脂质类似物材料为I2R2C18-1A1、I2R2C18-2A1、I2R3C18-1A1、I2R3C18-2A1中的至少一种。本发明在A549细胞(A549-Luc)检测阳离子脂质类似物材料的siRNA转染与递送效果，上述4种材料均可递送siRNA，能够实现高效的基因沉默。
进一步地，本发明所用的核酸，对其种类或结构没有特殊的限定，所述核酸包括但不限于信使RNA(mRNA)、小干扰RNA(siRNA)、短发夹RNA(shRNA)、微RNA(miRNA)、指导RNA(gRNA)、CRISPRRNA(crRNA)、反式激活RNA(tracrRNA)、质粒DNA(pDNA)、小环DNA、基因组DNA(gNDA)的至少一种。本发明所用的核酸，可以是来自人、动物、植物、细菌、病毒等的核酸，也可以是通过化学合成制备的核酸。进而，上述核酸可以是单链、双链、三链中的任一种，并且对其分子量也没有特殊的限定。此外，本发明的核酸可以为被化学、酶或肽修饰的核酸。
本发明还提供了上述阳离子脂质类似物材料的制备方法，具体方法为：将醛类化合物和胺类化合物加入到有机溶液中，反应10-120min后依次加入羧酸类化合物和异氰化合物，并于4-60℃反应6-72h，反应结束后经层析色谱柱分离提纯产物，得到所述阳离子脂质类似物材料。
本发明采用醛类化合物、胺类化合物、羧酸类化合物和异氰化合物为原料，通过Ugi反应合成小分子阳离子脂质类似物材料。本发明所述阳离子脂质类似物材料的反应条件温和，合成工艺简单、稳定性好，无需复杂的反应装置，合成的小分子阳离子脂质类似物材料毒性低，转染效率高，可将核酸药物高效递送到细胞内。
进一步地，在所述阳离子脂质类似物材料的制备方法中，采用甲醇和二氯甲烷的混合液作为流动相的条件下进行色谱柱分离。
进一步地，在所述阳离子脂质类似物材料的制备方法中，所述醛类化合物、胺类化合物、羧酸类化合物和异氰化合物的摩尔比为0.1-1:0.1-1:0.1-1:0.1-1；更进一步地，摩尔比为1:1:1:0.5。
进一步地，在所述阳离子脂质类似物材料的制备方法中，所述醛类化合物选自如下化合物A1-A3中的任一种：
Figure PCTCN2021136192-appb-000013
更进一步地，所述醛类化合物优选为A1化合物；
所述胺类化合物选自如下化合物R1-R11中的任一种：
Figure PCTCN2021136192-appb-000014
所述羧酸类化合物选自如下化合物CHS、C18-1、C18-2或C8-C20中的任一种：
Figure PCTCN2021136192-appb-000015
所述异氰化合物选自如下化合物I1、I2-1、I2、I2-3、I3中的任一种：
Figure PCTCN2021136192-appb-000016
与现有技术相比，本发明的有益效果为：
本发明的阳离子脂质类似物材料可以高效结合质粒DNA、mRNA、siRNA等，且其在多种细胞上可递送不同的质粒DNA，均具有较高的转染效率，甚至达到或高于目前商业化转染试剂的水平。本发明的阳离子脂质类似物材料可作为安全、高效的核酸药物胞内递送载体或转染试剂，具有实际的生物医学应用价值。
说明书附图
图1为阳离子脂质类似物材料I2-1R2C18A1的质谱(a)和核磁共振氢谱(b)。
图2为阳离子脂质类似物材料I2R2C18A1的质谱(a)和核磁共振氢谱(b)。
图3为阳离子脂质类似物材料I2-3R2C18A1的质谱(a)和核磁共振氢谱(b)。
图4为不同阳离子脂质类似物材料转染表达GFP的质粒DNA的阳性率结果。其中I2R11C14A1和I2R11C18-2A1的剂量为0.5微克/孔；I1R2C14A1、I2R1C16A1、I2R1C18A1、I2R1C18-1A1、I2R1C18-2A1、I2R2C18-2A1、I2R3C14A1、I2R3C16A1和I2R3C18-2A1的剂量为1微克/孔；I1R2C16A1、I1R2C18-1A1、I1R2C18-2A1、I1R3C14A1、I1R3C16A1、I1R3C18-2A1、I1R11C14A1、I1R11C16A1、I1R11C18A1、I1R11C18-1A1、I1R11C18-2A1、I2R1C14A1、I2R2C14A1、I2R2C16A1、I2R2C18-1A1、I2R3C18A1、I2R3C18-1A1和I2R11C16A1的剂量为2微克/孔；I1R1C16A1、I1R2C18A1、I1R3C12A1、I1R3C18A1、I1R3C18-1A1、I1R11C12A1、I2R2C18A1、I2R11C12A1、I2R11C18A1和I2R11C18-1A1的剂量为4微克/孔；I1R1C12A1、I1R1C14A1、I1R1C18A1、I1R1C18-1A1、I1R1C18-2A1、I1R2C12A1、I1R5C12A1、I1R5C14A1、I1R5C16A1、I1R5C18A1、I1R5C18-1A1、I1R5C18-2A1、I2R1C12A1、I2R2C12A1、I2R3C12A1、I2R5C12A1、I2R5C14A1、I2R5C16A1、I2R5C18A1、I2R5C18-1A1和I2R5C18-2A1的剂量为8微克/孔。
图5为不同阳离子脂质类似物材料转染表达荧光素酶的质粒DNA后，HeLa细胞的荧光素酶表达量的检测结果。
图6为不同添加量的I2R3C18-1A1转染表达GFP的质粒DNA至不同类型细胞的激光共聚焦结果。
图7为不同阳离子脂质类似物材料转染eGFP-mRNA的阳性率结果。其中I1R2C14A1、I1R2C18-1A1、I1R2C18-2A1、I1R11C14A1、I2R2C14A1、I2R2C18-1A1、I2R2C18-2A1、I2R3C16A1、I2R3C18A1、 I2R3C18-1A1、I2R3C18-2A1、I2R11C14A1、I2R11C16A1、I2R11C18-1A1和I2R11C18-2A1的剂量为1微克/孔；I1R1C12A1、I1R1C14A1、I1R1C16A1、I1R1C18A1、I1R1C18-1A1、I1R1C18-2A1、I1R2C12A1、I1R2C16A1、I1R2C18A1、I1R3C12A1、I1R3C14A1、I1R3C16A1、I1R3C18A1、I1R3C18-1A1、I1R3C18-2A1、I1R5C12A1、I1R5C14A1、I1R5C16A1、I1R5C18A1、I1R5C18-1A1、I1R5C18-2A1、I1R11C12A1、I1R11C16A1、I1R11C18A1、I1R11C18-1A1、I1R11C18-2A1、I2R1C12A1、I2R1C14A1、I2R1C16A1、I2R1C18A1、I2R1C18-1A1、I2R1C18-2A1、I2R2C12A1、I2R2C16A1、I2R2C18A1、I2R3C12A1、I2R3C14A1、I2R5C12A1、I2R5C14A1、I2R5C16A1、I2R5C18A1、I2R5C18-1A1、I2R5C18-2A1、I2R11C12A1和I2R11C18A1的剂量为2微克/孔。
图8为不同阳离子脂质类似物材料转染eGFP-mRNA的平均荧光强度。其中，实验条件与图7保持一致。
图9为I2R2C18-2A1、I2R3C18-2A1、I2R11C18-2A1转染eGFP-mRNA至DC2.4细胞的激光共聚焦结果，商品化转染试剂Lipofectamine 2000作为阳性对照。
图10为不同添加量的I2R2C18-1A1、I2R3C18-1A1、I2R2C18-2A1、I2R3C18-2A1转染siRNA的基因沉默结果。
图11为I2R2C16A1、I2R2C17A1、I2R2C18A1、I2R2C19A1、I2R2C20A1的细胞毒性结果。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明进一步说明。本领域技术人员应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
实施例中，所使用的实验方法如无特殊说明，均为常规方法，所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1 阳离子脂质类似物材料的合成与表征
本发明的阳离子脂质类似物材料的合成路线为：
Figure PCTCN2021136192-appb-000017
其中，胺类化合物m 2-NH 2为如下化合物R1-R11中的任一种；羧酸类化合物
Figure PCTCN2021136192-appb-000018
为如下化合物C8-C20、CHS中的任一种；醛类化合物
Figure PCTCN2021136192-appb-000019
为如下化合物A1-A3中的任一种；异氰化合物
Figure PCTCN2021136192-appb-000020
为如下化合物I1-I3中的任一种；
Figure PCTCN2021136192-appb-000021
本实施例的阳离子脂质类似物材料的具体制备方法为：分别将1mmol的异丁醛和1mmol的胺类化合 物加入到0.5mL甲醇溶液中，反应60min后依次加入1mmol羧酸类化合物和0.5mmol异氰化合物，并于40℃反应12h，反应结束后经层析色谱柱分离提纯产物，其中，流动相采用甲醇和二氯甲烷的混合液。
本实施例采用的原料及合成的阳离子脂质类似物材料结构如表1所示。
表1
Figure PCTCN2021136192-appb-000022
Figure PCTCN2021136192-appb-000023
Figure PCTCN2021136192-appb-000024
Figure PCTCN2021136192-appb-000025
Figure PCTCN2021136192-appb-000026
Figure PCTCN2021136192-appb-000027
Figure PCTCN2021136192-appb-000028
Figure PCTCN2021136192-appb-000029
Figure PCTCN2021136192-appb-000030
Figure PCTCN2021136192-appb-000031
Figure PCTCN2021136192-appb-000032
Figure PCTCN2021136192-appb-000033
Figure PCTCN2021136192-appb-000034
Figure PCTCN2021136192-appb-000035
Figure PCTCN2021136192-appb-000036
Figure PCTCN2021136192-appb-000037
Figure PCTCN2021136192-appb-000038
Figure PCTCN2021136192-appb-000039
选取阳离子脂质类似物I2-1R2C18A1、I2R2C18A1和I2-3R2C18A1作为表达材料，并对其结构进行表征。其中，I2-1R2C18A1的质谱和核磁共振氢谱的谱图如图1所示；I2R2C18A1的质谱和核磁共振氢谱的谱图如图2所示；I2-3R2C18A1的质谱和核磁共振氢谱的谱图如图3所示。核磁共振氢谱和质谱的结果与预期阳离子脂质类似物材料的结构一致。
实施例2 表达绿色荧光蛋白(GFP)的质粒DNA转染实验
本实验使用表达绿色荧光蛋白(GFP)的质粒DNA作为报告基因，在HeLa细胞检测阳离子脂质类似物材料的基因转染效率，具体方法如下：
将HeLa细胞接种在24孔板上并在细胞培养箱中培养12小时，将不同的阳离子脂质类似物材料(0.25-8微克/孔)和表达GFP的质粒DNA(0.5微克/孔)在40微升醋酸钠缓冲液(25mM，pH 5.2)中混合后静置10分钟，再稀释到460微升Opti-MEM培养基中，即可得到负载质粒DNA的阳离子脂质类似物复合物颗粒溶液。将HeLa细胞的培养基去掉，使用PBS清洗一遍后加入复合好的颗粒溶液，培养24小时后采用流式细胞仪分析细胞内的质粒DNA转染效率。以商业化基因转染试剂Lipofectamine 2000作为阳性对照。
由图4结果可知，本发明的阳离子脂质类似物材料能够转染表达GFP的质粒DNA至HeLa细胞，尤其是I1R2C14A1、I1R2C18-2A1、I1R11C14A1、I2R1C14A1、I2R1C16A1、I2R1C18-1A1、I2R1C18-2A1、I2R2C14A1、I2R2C16A1、I2R2C18-1A1、I2R2C18-2A1、I2R3C16A1、I2R3C18A1、I2R3C18-1A1、I2R3C18-2A1、I2R11C14A1、I2R11C16A1、I2R11C18A1、I2R11C18-1A1、I2R11C18-2A1的转染效率能 够达到或高于商品化转染试剂Lipofectamine 2000。
实施例3 表达荧光素酶(luminescence)的质粒DNA转染实验
本实施例以表达荧光素酶的报告基因作为模型质粒DNA，在HeLa细胞检测阳离子脂质类似物材料的基因转染效率，具体操作方法如下：将HeLa细胞接种在96孔板上并在细胞培养箱中培养12小时，分别将不同的阳离子脂质类似物材料和表达荧光素酶的质粒DNA(0.5微克)在40微升醋酸钠缓冲液(25mM，pH 5.2)中混合后静置10分钟，再稀释到460微升Opti-MEM培养基中，即可得到负载质粒DNA的阳离子脂质类似物复合物颗粒溶液。将HeLa细胞的培养基去掉，使用PBS清洗一遍后加入125微升复合好的颗粒溶液。培养24小时后，弃掉培养溶液，充分裂解后加入50微升/孔的底物，使用多功能酶标仪检测荧光素酶的表达量。
由图5结果可知，I2R3C18A1、I2R1C18-1A1、I1R11C14A1、I2R11C18-2A1、I1R2C14A1、I2R1C14A1、I2R1C16A1、I2R3C18-2A1、I2R2C18-2A1、I2R2C16A1、I2R3C16A1、I2R2C18-1A1、I2R3C18-1A1的转染效率能够达到或高于商品化转染试剂Lipofectamine 2000。
实施例4 I2R3C18-1A1转染表达GFP的质粒DNA至不同类型细胞的效果
本实验以I2R3C18-1A1作为代表性的阳离子脂质类似物材料，以表达GFP的质粒DNA作为报告基因，探究不同添加量的阳离子脂质类似物材料(0.5-3微克/孔)在不同类型细胞内的质粒DNA(0.5微克/孔)转染效果。以商业化基因转染试剂Lipofectamine 2000作为阳性对照。本实验参照实施例8的方法制备负载表达GFP的质粒DNA的I2R3C18-1A1颗粒溶液，分别在小鼠树突状细胞(DC 2.4)、小鼠巨噬细胞(RAW264.7)、腺癌人类肺泡基底上皮细胞(A549)、人胰腺癌细胞(BxPC3)和HeLa细胞中加入负载表达GFP的质粒DNA的I2R3C18-1A1颗粒溶液复合物溶液，培养24小时后，采用激光共聚焦显微镜观察细胞内的质粒DNA转染效率。
图6结果表明，I2R3C18-1A1材料可转染表达GFP的质粒DNA至肿瘤细胞以及免疫细胞。
实施例5 表达增强绿色荧光蛋白(eGFP)mRNA转染实验
本实验选用eGFP-mRNA作为模型mRNA，在DC 2.4细胞检测阳离子脂质类似物材料的mRNA转染效率，具体操作方法如下：
将DC 2.4细胞接种在48孔板上并在细胞培养箱中培养12小时，将不同的阳离子脂质类似物材料(0.25–2微克/孔)和表达eGFP的mRNA(0.2微克/孔)在20微升醋酸钠缓冲液(25mM，pH 5.2)中混合后静置10分钟，再稀释到230微升Opti-MEM培养基中，即可得到负载mRNA的复合颗粒。将HeLa细胞的培养基去掉，使用PBS清洗一遍后加入复合好的颗粒溶液，培养24小时后采用流式细胞仪以及激光共聚焦显微镜观察细胞内的mRNA转染效率。以商业化基因转染试剂Lipofectamine 2000作为阳性对照。
图7～9结果显示，本发明的阳离子脂质类似物材料可将表达eGFP的mRNA转染至DC 2.4细胞，尤其I1R2C14A1、I1R2C16A1、I1R2C18A1、I1R2C18-1A1、I1R2C18-2A1、I1R11C14A1、I1R11C16A1、I1R11C18A1、I2R1C14A1、I2R1C16A1、I2R1C18-1A1、I2R2C14A1、I2R2C16A1、I2R2C18A1、I2R2C18-1A1、I2R2C18-2A1、I2R3C14A1、I2R3C16A1、I2R3C18A1、I2R3C18-1A1、I2R3C18-2A1、I2R11C14A1、I2R11C16A1、I2R11C18A1、I2R11C18-1A1、I2R11C18-2A1的mRNA转染效率可达到或高于商品化转染试剂Lipofectamine 2000。
实施例6 小干扰核酸(siRNA)转染实验
本实验选择了I2R2C18-1A1、I2R3C18-1A1、I2R2C18-2A1、I2R3C18-2A1作为代表性的阳离子脂质类似物材料，在A549细胞(A549-Luc)检测阳离子脂质类似物材料的siRNA转染与递送效果，具体操作方法如下：
将表达荧光素酶的A549细胞(A549-Luc)接种在96孔板上并在细胞培养箱中培养12小时，将不同的材料(0.5–3微克/孔)和siRNA在40微升醋酸钠缓冲液(25mM，pH 5.2)中混合后静置10分钟，再稀释到460微升Opti-MEM培养基中，即可得到负载siRNA的复合颗粒(siRNA最终浓度为100nM)；将A549-Luc细胞的培养基去掉，使用PBS清洗一遍后加入125微升复合好的颗粒溶液；培养48小时后，弃掉培养溶液，充分裂解后加入50微升/孔的底物，使用多功能酶标仪检测荧光素酶的表达量。
图10结果表明，I2R2C18-1A1、I2R3C18-1A1、I2R2C18-2A1、I2R3C18-2A1可将siRNA转染到细胞中，特异性地沉默荧光素酶报告基因的表达，且随着阳离子脂质类似物材料添加量增加，基因沉默效率提高。
实施例7 阳离子脂质类似物材料的细胞毒性测试
本实验选择了转染效率较高的I2R2C16A1、I2R2C18A1以及I2R2C17A1、I2R2C19A1、I2R2C20A1作为代表性的阳离子脂质类似物材料，用MTT实验检测阳离子脂质类似物材料对HeLa细胞的毒性，具体实验方法为：HeLa细胞铺在96孔板上并在细胞培养箱中培养12小时，随后将细胞培养基去掉，换成1微克/毫升的阳离子类脂质材料并培养4小时，然后将材料洗掉，并换成DMEM培养基培养20小时，最后使用MTT检测细胞存活率。
图11结果表明，本发明的阳离子脂质类似物材料I2R2C16A1、I2R2C17A1、I2R2C18A1、I2R2C19A1、I2R2C20A1细胞毒性较小，具有良好的生物相容性。
此外，发明人在前期的研究中发现，将上述实施例1中的异丁醛替换为
Figure PCTCN2021136192-appb-000040
时，所制备得到的阳离子脂质类似物材料也具有细胞毒性低的特点，使用表达绿色荧光蛋白(GFP)的质粒DNA或者表达荧光素酶(luminescence)的质粒DNA为报告基因，在HeLa细胞检测阳离子脂质类似物材料的基因转染效率，这些材料也具有一定效果。因此，可以推测这些阳离子脂质类似物材料也可以作为核酸药物递送载体。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (10)

  1. 可电离的阳离子脂质类似物材料在作为核酸药物递送载体或转染试剂中的应用，其特征在于，所述阳离子脂质类似物材料具有如式(I)所示结构：
    Figure PCTCN2021136192-appb-100001
    式(I)中，m 1独立地选自直链烷基、支链烷基、苯基或含杂原子的芳香基；
    m 2为
    Figure PCTCN2021136192-appb-100002
    R 1为烷基，R 2为烷基，R 3为烷基或苯基，或R 2与R 3连接为环基或杂环基；
    m 3独立地选自直链烷基、直链烯基或
    Figure PCTCN2021136192-appb-100003
    m 4独立地选自直链烷基、含醚键的直链烷基或含有含N杂环的烷基。
  2. 根据权利要求1所述的应用，其特征在于，m 1为被取代基α取代的烷基、苯基或含杂原子的芳香基，所述取代基包括甲基，优选地，m 1为
    Figure PCTCN2021136192-appb-100004
  3. 根据权利要求1所述的应用，其特征在于，m 2为
    Figure PCTCN2021136192-appb-100005
    Figure PCTCN2021136192-appb-100006
  4. 根据权利要求1所述的应用，其特征在于，m 3为碳原子数为7-19的直链烷基、碳原子数为17的直链烯基或
    Figure PCTCN2021136192-appb-100007
    优选地，m 3为
    Figure PCTCN2021136192-appb-100008
    Figure PCTCN2021136192-appb-100009
  5. 根据权利要求1所述的应用，其特征在于，m 4为碳原子数为6的直链烷基、碳原子数为4-8的含醚键的直链烷基或含有含N杂环的烷基，优选地，m 4为
    Figure PCTCN2021136192-appb-100010
    Figure PCTCN2021136192-appb-100011
  6. 根据权利要求1所述的应用，其特征在于，所述阳离子脂质类似物材料具有如下72种结构中的任一种：
    Figure PCTCN2021136192-appb-100012
    Figure PCTCN2021136192-appb-100013
  7. 根据权利要求6所述的应用，其特征在于，所述阳离子脂质类似物材料为I1R2C14A1、I1R2C18-2A1、I1R11C14A1、I2R1C14A1、I2R1C16A1、I2R1C18-1A1、I2R1C18-2A1、I2R2C14A1、I2R2C16A1、I2R2C18-1A1、I2R2C18-2A1、I2R3C16A1、I2R3C18A1、I2R3C18-1A1、I2R3C18-2A1、I2R11C14A1、I2R11C16A1、I2R11C18A1、I2R11C18-1A1、I2R11C18-2A1中的至少一种。
  8. 根据权利要求6所述的应用，其特征在于，所述阳离子脂质类似物材料为I1R2C14A1、I1R2C16A1、 I1R2C18A1、I1R2C18-1A1、I1R2C18-2A1、I1R11C14A1、I1R11C16A1、I1R11C18A1、I2R1C14A1、I2R1C16A1、I2R1C18-1A1、I2R2C14A1、I2R2C16A1、I2R2C18A1、I2R2C18-1A1、I2R2C18-2A1、I2R3C14A1、I2R3C16A1、I2R3C18A1、I2R3C18-1A1、I2R3C18-2A1、I2R11C14A1、I2R11C16A1、I2R11C18A1、I2R11C18-1A1、I2R11C18-2A1中的至少一种。
  9. 根据权利要求6所述的应用，其特征在于，所述阳离子脂质类似物材料为I2R2C18-1A1、I2R2C18-2A1、I2R3C18-1A1、I2R3C18-2A1中的至少一种。
  10. 根据权利要求1-9任一项所述的应用，其特征在于，所述核酸包括信使RNA、小干扰RNA、短发夹RNA、微RNA、指导RNA、CRISPR RNA、反式激活RNA、质粒DNA、小环DNA、基因组DNA的至少一种。
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