WO2023142167A1 - 一种阳离子脂质类似物、其组合物及应用 - Google Patents

一种阳离子脂质类似物、其组合物及应用 Download PDF

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WO2023142167A1
WO2023142167A1 PCT/CN2022/075911 CN2022075911W WO2023142167A1 WO 2023142167 A1 WO2023142167 A1 WO 2023142167A1 CN 2022075911 W CN2022075911 W CN 2022075911W WO 2023142167 A1 WO2023142167 A1 WO 2023142167A1
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cationic lipid
compound
heteroatom
drug
straight
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PCT/CN2022/075911
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French (fr)
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刘志佳
陈永明
贺泽芃
乐志成
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广州立得生物医药科技有限公司
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Priority to EP22922990.1A priority Critical patent/EP4461316A1/en
Publication of WO2023142167A1 publication Critical patent/WO2023142167A1/zh

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Definitions

  • the invention relates to the technical field of biomedicine, in particular to a cationic lipid analogue, its composition and application.
  • Liposomes are completely closed multilayered vesicles with bilayers formed by lipids such as phospholipids, which can encapsulate water-soluble and fat-soluble substances, and have the characteristics of high targeting efficiency, controlled release, safety and non-toxicity .
  • lipids such as phospholipids
  • liposomes and their analogues have become more and more mature, and more and more studies have shown the potential of various lipid nanoparticles in molecular drug therapy.
  • liposomes can be divided into cationic liposomes, neutral liposomes and anionic liposomes.
  • cationic liposomes have the advantages of being naturally degradable, non-toxic and non-immunogenic, and capable of repeated transfection.
  • Cationic liposomes are usually used as gene carriers, complexed with DNA or RNA, to protect them from being inactivated or degraded by nucleases, carrying genes that can be transported to specific parts, and the transfection process must be convenient and reproducible good.
  • the liposome delivery system with high gene delivery efficiency can realize the appropriate expression of specific genes in specific organs in the organism to achieve therapeutic applications.
  • the gene therapy applications that have been developed in the prior art include nucleic acid therapy, nucleic acid vaccines, protein replacement therapy, Gene editing and more.
  • nucleic acid vaccines include nucleic acid vaccines, protein replacement therapy, Gene editing and more.
  • messenger RNA vaccines have attracted more attention due to their ease of preparation, high efficiency and stability.
  • the purpose of the present invention is to overcome the deficiencies of the prior art and provide a cationic lipid analogue, its composition and application.
  • the present invention prepares a cationic lipid analog, which has a structure as shown in any one of formula (I) to formula (V):
  • m is independently selected from a hydrogen atom, a straight-chain alkyl group, a branched-chain alkyl group, a straight-chain alkenyl group, a branched chain alkenyl group, a substituted alkynyl group, a cycloalkyl group, a phenyl group, and a heteroatom-containing aromatic group at least one group of
  • m2 is independently selected from straight-chain alkyl, straight-chain alkenyl, heteroatom-containing straight-chain alkyl, heteroatom-containing straight-chain alkylene, heteroatom-containing branched-chain alkyl, heteroatom-containing branched-chain alkylene , an alkyl group containing a heteroatom ring, an alkylene group containing a heteroatom ring, an alkyl group containing an ester group, and an alkyl group containing an ether group;
  • m3 is independently selected from straight-chain alkyl, straight-chain alkenyl, cycloalkyl, heteroatom-containing straight-chain alkyl, heteroatom-containing branched-chain alkyl, heteroatom-containing branched-chain alkylene, heteroatom-containing ring At least one of alkyl groups, alkyl groups containing heteroatoms and aromatic rings, alkyl groups containing hydroxyl groups, alkyl groups containing ester groups, alkyl groups containing ether groups, and alkyl groups containing sulfonate groups;
  • m4 is independently selected from straight-chain alkyl, branched-chain alkyl, straight-chain alkenyl, branched alkenyl, straight-chain alkynyl, heteroatom-containing straight-chain alkyl, heteroatom-containing branched-chain alkyl, heteroatom-containing At least one group in the alkyl group of the atomic ring.
  • the present invention uses aldehyde compounds, amine compounds, carboxylic acid compounds and isonitrile compounds as raw materials to synthesize cationic lipid analogs through Ugi reaction; cationic lipid analogs have high in vivo transfection efficiency, low toxicity and specific targeting It can achieve mRNA expression in different organs of animals through different administration methods, and can meet the application requirements of mRNA nucleic acid therapy, nucleic acid vaccine and gene editing, etc.
  • m 1 is independently selected from and / or
  • n 2 is independently selected from and/or m 3 are independently selected from and / or
  • n is independently selected from
  • the m 1 is and / or
  • the m2 is, and / or
  • the m3 is and / or
  • the m 4 is
  • the present invention provides a method for preparing the cationic lipid analogue, adding aldehyde compounds and amine compounds into an organic solution, and adding carboxylic acid compounds and isocyanides in sequence after reacting for 10 to 120 minutes compound, react at 4-60°C for 6-72 hours, separate and purify the product, and it is ready.
  • the molar ratio of the aldehyde compounds, amine compounds, carboxylic acid compounds and isonitrile compounds is (0.1-1.5): (0.1-1.5): (0.1-1.5); further Generally, the molar ratio is 1:(0.33 ⁇ 1):1:(0.33 ⁇ 1).
  • reaction condition of the cationic lipid analog of the present invention is mild, the synthesis process is simple, and the stability is good.
  • the isonitrile compound is one of monofunctional isonitrile compounds, bifunctional isonitrile compounds, and trifunctional isonitrile compounds.
  • the isonitrile compound is any one of the following compounds I3-I38:
  • the aldehyde compound is one of monofunctional aldehyde compounds.
  • the aldehyde compound is any one of the following compounds A1-A35:
  • the amine compound is one of monofunctional amine compounds, bifunctional amine compounds, and trifunctional amine compounds.
  • the amine compound is any one of the following compounds R1-R53:
  • the carboxylic acid compound is any one of the following compounds C8-C30 or S11-S19:
  • the present invention provides a composition of the cationic lipid analog, including at least one of the cationic lipid analog and sterol, auxiliary lipid, polyethylene glycol lipid derivative kind.
  • the sterols include at least one of cholesterol, sitosterol (sitosterol), stigmasterol (stigmasterol), and cholesterol derivatives;
  • the auxiliary lipid is a non-cationic lipid, more preferably, the auxiliary lipid is a neutral phospholipid, further, the auxiliary lipid is phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide at least one.
  • the molar ratio of the cationic lipid analogs, cholesterol, helper lipids, and polyethylene glycol lipid derivatives is (20-70): (20-50): (2-30): (0.1-20); preferably, the molar ratio is (40-60): (25-45): (5-15): (0.5-5).
  • the present invention provides a drug-loaded nanoparticle, comprising the composition and the drug of the cationic lipid analog.
  • the drug includes at least one of small molecular compounds, nucleic acid molecules, protein or polypeptide molecules, and gene editing complexes.
  • the nucleic acid molecule is at least one of messenger RNA (mRNA), transfer RNA (tRNA), dsRNA, shRNA, DNA, plasmid DNA, siRNA, antisense oligonucleotides, aiRNA, miRNA; the gene
  • the editing complex is mRNA/sgRNA or Cas9/sgRNA.
  • the present invention provides a method for preparing the drug-loaded nanoparticles, comprising the following steps:
  • the present invention uses the cationic lipid analogs, the composition of the cationic lipid analogs, and the drug-loaded nanoparticles in the preparation, delivery or delivery of molecular drugs or nucleic acid vaccines.
  • the present invention uses aldehyde compounds, amine compounds, carboxylic acid compounds and isonitrile compounds as raw materials to synthesize cationic lipid analogs through Ugi reaction; the reaction conditions of the cationic lipid analogs of the present invention are mild, and the synthesis process is simple and stable Good; synthetically obtained cationic lipid analogs form lipid nanoparticle-like compositions with sterols, helper lipids, and polyethylene glycol lipid derivatives, which can be used for drug delivery, including small molecule compounds, mRNA, DNA, siRNA and other nucleic acid molecules, protein/polypeptide molecules, and mRNA/sgRNA, Cas9/sgRNA and other gene editing complexes; through different drug delivery methods to achieve mRNA expression in different organs in animals, which can meet the needs of mRNA nucleic acid therapy and nucleic acid vaccines And application requirements such as gene editing.
  • Fig. 1 is the proton nuclear magnetic resonance spectrogram of A4I18R3C18-2 in embodiment 1.
  • FIG. 2 is the mass spectrum of A4I18R3C18-2 in Example 1.
  • Fig. 3 is the proton nuclear magnetic resonance spectrogram of A1I4R22C18-2 in embodiment 2.
  • Fig. 4 is the mass spectrogram of A1I4R22C18-2 in embodiment 2.
  • Fig. 5 is the proton nuclear magnetic resonance spectrogram of A1I5R22S15 in embodiment 3.
  • Fig. 6 is the mass spectrogram of A1I5R22S15 in embodiment 3.
  • Figure 7 is the H NMR spectrum of A1I32R46C18-2 in Example 4.
  • FIG. 8 is the mass spectrum of A1I32R46C18-2 in Example 4.
  • FIG. 9 is the H NMR spectrum of A1I32R47C18-2 in Example 5.
  • FIG. 10 is the mass spectrum of A1I32R47C18-2 in Example 5.
  • Fig. 11 is a statistical chart of antibody titers after immunization with OVA-mRNA drug-loaded nanoparticles prepared by the compound of Example 1.
  • Fig. 12 is a statistical chart of antibody titers after immunization with OVA-mRNA drug-loaded nanoparticles prepared by the compound of Example 2.
  • test methods used in the examples are conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources.
  • Embodiment 1 A kind of cationic lipid analogue
  • the specific preparation method is:
  • the aldehyde compound is any one of A1, A4, A23; the isonitrile compound is I18; the amine compound is any one of R1, R2, R3; the carboxylic acid compound is C18, C18-1, C18- 2. Any one of C18-3, C23.
  • the cationic lipid analogs synthesized by the above-mentioned preferred compounds are the following 45 kinds:
  • the aldehyde compound is any one of A1, A4, A23; the isonitrile compound is I18; the amine compound is any one of R5, R12; the carboxylic acid compound is any one of C18, C18-2.
  • the cationic lipid analogs synthesized by the above-mentioned preferred compounds are the following 12 kinds:
  • the aldehyde compound is A4 or A5; the isonitrile compound is I10, I14 or I18; the amine compound is R4, R6, R7, R8, R9, R10 or R11; the carboxylic acid compound is C18 or C18-2.
  • the cationic lipid analogs synthesized by the above-mentioned preferred compounds are the following 10 kinds:
  • Embodiment 2 A kind of cationic lipid analog
  • the specific preparation method is:
  • the aldehyde compound is any one of A1, A4, A21, A23; the isonitrile compound is I4; the amine compound is any one of R2, R13, R21, R22, R23; the carboxylic acid compound is C18, Either of C18-1, C18-2.
  • the cationic lipid analogues synthesized by the above compounds are the following 60 kinds:
  • Aldehyde compound is A1; isonitrile compound is I4; amine compound is any one of R27, R28, R29, R30, R31, R32, R33, R34, R35, R36, R42, R43, R48; carboxylic acid The compound is C18-2.
  • the cationic lipid analogues synthesized by the above compounds are the following 13 kinds:
  • the aldehyde compound is any one of A1; the isonitrile compound is I4; the amine compound is R22; the carboxylic acid compound is any one of C12, C14, C16, S11, S13, S15, S17, S19.
  • Adopt the synthetic cationic lipid analog of above-mentioned compound to be following 8 kinds:
  • Embodiment 3 A kind of cationic lipid analog
  • the specific preparation method is:
  • the aldehyde compound is A1; the isonitrile compound is I5; the amine compound is R2, R13, R21, R22, R23, R26, R27, R28, R29, R30, R31, R32, R33, R34, R35, R36, R37, R38, R39, R40, R41 or R50; carboxylic acid compounds are C8, C10, C12, C14, C16, C18, C18-2, C21, S11, S13, S15, S17 or S19.
  • the cationic lipid analogues synthesized by the above compounds are the following 29 kinds:
  • Embodiment 4 A kind of cationic lipid analog
  • the specific preparation method is:
  • the aldehyde compound is A1; the isonitrile compound is I10, I12, I31 or I32; the amine compound is R46 or R51; the carboxylic acid compound is C8 or C18-2.
  • Adopt the synthetic cationic lipid analog of above-mentioned compound to be following 4 kinds:
  • A1I12R51C8 A1I10R51C18-2, A1I31R46C18-2, A1I32R46C18-2.
  • Embodiment 5 A kind of cationic lipid analog
  • the specific preparation method is:
  • the aldehyde compound is A1; the isonitrile compound is I12, I31 or I32; the amine compound is R47; the carboxylic acid compound is C21 or C18-2.
  • Adopt the synthetic cationic lipid analogue of above-mentioned compound to be following 3 kinds:
  • Embodiment 6 A kind of drug-loaded nanoparticles
  • the drug-loaded nanoparticles include cationic lipid analogs, cholesterol, distearoylphosphatidylcholine (DSPC), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG -PEG).
  • DSPC distearoylphosphatidylcholine
  • DMG -PEG 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol
  • Table 1 uses embodiment 6 to prepare lipid nanoparticle characterization data
  • Embodiment 7 A kind of drug-loaded nanoparticles
  • the drug-loaded nanoparticles include cationic lipid analogs, ⁇ -sitosterol, dioleoylphosphatidylethanolamine (DOPE), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG).
  • DOPE dioleoylphosphatidylethanolamine
  • DMG-PEG 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol
  • Embodiment 8 A kind of drug-loaded nanoparticles
  • the drug-loaded nanoparticles include cationic lipid analogs, stigmasterol, ceramide phosphorylcholine, and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG).
  • Embodiment 9 A kind of drug-loaded nanoparticles
  • the drug-loaded nanoparticles include cationic lipid analogs, cholesterol succinate monoester (cholesterol derivative), sphingomyelin, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG -PEG).
  • cationic lipid analogue:cholesterol succinate monoester:sphingomyelin:DMG-PEG 60:45:15:3.5.
  • Example 10 Animal experiments to detect the expression level of mRNA delivered in vivo by intravenous injection
  • Luciferase-mRNA was used as the model mRNA; the cationic lipid analogs of Examples 1-5 were used to prepare drug-loaded nanoparticles according to the method of Example 6.
  • Example 2 prepares the in vivo expression of mRNA drug-loaded nanoparticles intravenously injected
  • Example 3 prepares the in vivo expression of mRNA drug-loaded nanoparticles intravenously injected
  • Example 4 prepares the in vivo expression of mRNA drug-loaded nanoparticles intravenously injected
  • Example 5 prepares the in vivo expression of mRNA drug-loaded nanoparticles intravenously injected
  • Example 11 Animal experiments to detect the expression level of mRNA delivered in vivo by intramuscular injection
  • Luciferase-mRNA was used as the model mRNA; the cationic lipid analogs of Examples 1-5 were used to prepare drug-loaded nanoparticles according to the method of Example 6.
  • Example 4 prepares the in vivo expression of mRNA drug-loaded nanoparticles intramuscularly injected
  • Example 12 Animal experiments to detect the expression level of mRNA delivered in vivo by intraperitoneal injection
  • Luciferase-mRNA was used as model mRNA; the cationic lipid analog A1I4R22C18-2 of Example 2 was used to prepare drug-loaded nanoparticles according to the method of Example 6.
  • Example 13 Animal experiments to detect the expression level of mRNA delivered in vivo by tracheal injection
  • Luciferase-mRNA was used as model mRNA; the cationic lipid analog A1I4R22C18-2 of Example 2 was used to prepare drug-loaded nanoparticles according to the method of Example 6.
  • Table 13 The in vivo expression of mRNA drug-loaded nanoparticles prepared by the compound of Example 2 for tracheal injection
  • Example 14 Animal experiments evaluate the immune performance of mRNA delivered in vivo
  • OVA-mRNA was used as the model mRNA; the cationic lipid analogs of Examples 1 and 2 were used to prepare drug-loaded nanoparticles according to the method of Example 6.
  • Example 1 Taking the cationic lipid analog in Example 1 as an example, after intramuscular injection of 5 ⁇ g of OVA-mRNA nanoparticles or the drug-loaded nanoparticles prepared above on the 0th and 14th days, blood was taken on the 21st day for antibody titer analysis , the result is shown in Figure 11.
  • Example 2 Taking the cationic lipid analog A1I4R22C18-2 in Example 2 as an example, after intramuscularly or intraperitoneally injecting 5 ⁇ g of OVA-mRNA nanoparticles or the drug-loaded nanoparticles prepared above on the 0th and 7th days, blood was collected on the 14th day Antibody titer analysis was carried out, and the results are shown in FIG. 12 .
  • Example 15 Animal experiments to detect the delivery level of siRNA in vivo by intravenous injection
  • Cy5-siRNA was used as the model siRNA; the cationic lipid analogs A4I18R2C18-2 and A1I4R22C18-2 in Example 1 were used to prepare drug-loaded nanoparticles according to the method in Example 6.
  • nanoparticles containing 10 ⁇ g Cy5-siRNA or the drug-loaded nanoparticles prepared above were injected intravenously into the legs of C57BL/6 mice aged 6-8 weeks, and the in vivo siRNA delivery was evaluated by bioluminescence imaging 1 hour later.

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Abstract

一种阳离子脂质类似物、其组合物及应用。采用醛类化合物、胺类化合物、羧酸类化合物和异腈化合物为原料，通过Ugi反应合成阳离子脂质类似物；得到的阳离子脂质类似物与固醇、辅助脂质和聚乙二醇脂质衍生物形成脂质纳米颗粒样组合物，该组合物可用于药物输送，包括小分子药物，mRNA、DNA、siRNA等核酸药物，蛋白/多肽类药物，以及mRNA/sgRNA,Cas9/sgRNA等基因编辑复合物；通过不同给药方式实现在动物体内不同器官的mRNA表达，可满足mRNA核酸疗法、核酸疫苗以及基因编辑等的应用需求。

Description

一种阳离子脂质类似物、其组合物及应用 技术领域
本发明涉及生物医药技术领域，具体涉及一种阳离子脂质类似物、其组合物及应用。
背景技术
脂质体是由磷脂等类脂形成的双分子层的完全封闭的多层囊泡，可包封水溶性和脂溶性两种物质，具有靶向高效、缓释可控和安全无毒的特点。近年来通过利用脂质体以及其类似物递送分子药物的技术越来越成熟，越来越多的研究表明了各种脂质纳米粒在分子药物治疗中的潜力。
按电荷性质，脂质体可分为阳离子脂质体、中性脂质体和阴离子脂质体。其中，阳离子脂质体具有可自然降解、无毒无免疫原性、可重复转染等优点。阳离子脂质体通常是用作基因载体，与DNA或RNA复合，保护其不被灭活或被核酸酶降解，携带基因可转运至特定部位，并且要保证转染过程方便易行，重现性好。通过高基因递送效率的脂质体递送系统可以实现特定基因在生物体内的特定器官适当表达，以实现治疗应用，现有技术中已经开发的基因治疗应用包括核酸治疗、核酸疫苗、蛋白替代治疗、基因编辑等等。特别的，随着核酸疫苗在近年来的快速发展，信使RNA疫苗凭借其制备简易型，高效性与稳定性而更加受到重视。
目前，阳离子脂质颗粒的研究已经多有报道，并且也有商品化的阳离子脂质材料可供选择，如阳离子磷脂DOTAP、可电离的阳离子脂质DLin-MC3，但仍存在体内递送效率不高，阳离子对于生物机体有毒性，体内递送靶向性不强等问题。
鉴于阳离子脂质类似物结构的细微差别就会导致其生物性能发生重大改变，因此，对于体内高转染效率、低毒性和特异靶向性的新型阳离子脂质类似物结构 的开发至关重要。
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一种阳离子脂质类似物、其组合物及应用。
为实现上述目的，本发明采取的技术方案如下：
第一方面，本发明制备了一种阳离子脂质类似物，其具有如式(I)～式(Ⅴ)任一种所示结构：
Figure PCTCN2022075911-appb-000001
Figure PCTCN2022075911-appb-000002
式中，m 1为独立地选自氢原子、直链烷基、支链烷基、直链烯基、支链烯基、取代炔基、环烷基、苯基、含杂原子芳香基中的至少一种基团；
m 2为独立地选自直链烷基、直链烯基、含杂原子直链烷基、含杂原子直链亚烷基、含杂原子支链烷基、含杂原子支链亚烷基、含杂原子环的烷基、含杂原子环的亚烷基、含酯基的烷基、含醚基的烷基中的至少一种基团；
m 3为独立地选自直链烷基、直链烯基、环烷基、含杂原子直链烷基、含杂原子支链烷基、含杂原子支链亚烷基、含杂原子环的烷基、含杂原子和芳香环的烷基、含羟基的烷基、含酯基的烷基、含醚基的烷基、含磺酸酯基的烷基中的至少一种基团；
m 4为独立地选自直链烷基、支链烷基、直链烯基、支链烯基、直链炔基、含杂原子直链烷基、含杂原子支链烷基、含杂原子环的烷基中的至少一种基团。
本发明采用醛类化合物、胺类化合物、羧酸类化合物和异腈化合物为原料，通过Ugi反应合成阳离子脂质类似物；阳离子脂质类似物体内转染效率高，具有低毒性和特异靶向性，可通过不同给药方式实现在动物体内不同器官的mRNA表达，可满足mRNA核酸疗法、核酸疫苗以及基因编辑等的应用需求。
作为本发明所述的阳离子脂质类似物的优选实施方式，式中，m 1为独立地选自
Figure PCTCN2022075911-appb-000003
Figure PCTCN2022075911-appb-000004
和/或
m 2为独立地选自
Figure PCTCN2022075911-appb-000005
Figure PCTCN2022075911-appb-000006
Figure PCTCN2022075911-appb-000007
和/或m 3为独立地选自
Figure PCTCN2022075911-appb-000008
Figure PCTCN2022075911-appb-000009
Figure PCTCN2022075911-appb-000010
Figure PCTCN2022075911-appb-000011
和/或
m 4为独立地选自
Figure PCTCN2022075911-appb-000012
Figure PCTCN2022075911-appb-000013
优选地，所述m 1为
Figure PCTCN2022075911-appb-000014
Figure PCTCN2022075911-appb-000015
和/或
所述m 2为、
Figure PCTCN2022075911-appb-000016
Figure PCTCN2022075911-appb-000017
Figure PCTCN2022075911-appb-000018
和/或
所述m 3为
Figure PCTCN2022075911-appb-000019
Figure PCTCN2022075911-appb-000020
Figure PCTCN2022075911-appb-000021
和/或
所述m 4为
Figure PCTCN2022075911-appb-000022
Figure PCTCN2022075911-appb-000023
第二方面，本发明提供了一种所述的阳离子脂质类似物的制备方法，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腈化合物，再于4～60℃反应6～72h，分离提纯产物，即成。
优选地，所述醛类化合物、胺类化合物、羧酸类化合物和异腈化合物的摩尔比为(0.1～1.5)：(0.1～1.5)：(0.1～1.5)：(0.1～1.5)；进一步地，摩尔比为1：(0.33～1)：1：(0.33～1)。
本发明阳离子脂质类似物的反应条件温和，合成工艺简单、稳定性好。
作为本发明所述的阳离子脂质类似物的制备方法的优选实施方式，所述异腈化合物为单官能团异腈化合物、双官能团异腈化合物、三官能团异腈化合物中的一种。
优选地，所述异腈化合物为如下化合物I3～I38中的任一种：
Figure PCTCN2022075911-appb-000024
作为本发明所述的阳离子脂质类似物的制备方法的优选实施方式，所述醛类化合物为单官能团醛类化合物中的一种。
优选地，所述醛类化合物为如下化合物A1～A35中的任一种：
Figure PCTCN2022075911-appb-000025
作为本发明所述的阳离子脂质类似物的制备方法的优选实施方式，所述胺类化合物为单官能团胺类化合物、双官能团胺类化合物、三官能团胺类化合物中的一种。
作为本发明所述的阳离子脂质类似物的制备方法的优选实施方式，所述胺类化合物为如下化合物R1～R53中的任一种：
Figure PCTCN2022075911-appb-000026
作为本发明所述的阳离子脂质类似物的制备方法的优选实施方式，羧酸类化合物为如下化合物C8～C30或S11～S19中的任一种：
Figure PCTCN2022075911-appb-000027
第三方面，本发明提供了一种所述的阳离子脂质类似物的组合物，包括所述阳离子脂质类似物和固醇、辅助脂质、聚乙二醇脂质衍生物中的至少一种。
作为本发明所述阳离子脂质类似物的组合物的优选实施方式，所述固醇包括胆固醇、谷固醇(谷甾醇)、豆甾醇(豆固醇)、胆固醇衍生物中的至少一种；所述辅助脂质为非阳离子脂质，更优选地，所述辅助脂质为中性磷脂，进一步地，所述辅助脂质为磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂、神经酰胺中的至少一种。
作为本发明所述阳离子脂质类似物的组合物的优选实施方式，所述阳离子脂质类似物、胆固醇、辅助脂质、聚乙二醇脂质衍生物的摩尔比为(20～70)：(20～50)：(2～30)：(0.1～20)；优选地，摩尔比为(40～60)：(25～45)：(5～15)：(0.5～5)。
第四方面，本发明提供一种载药纳米颗粒，包括所述的阳离子脂质类似物的 组合物和药物。
作为本发明所述载药纳米颗粒的优选实施方式，所述药物包括小分子化合物，核酸分子，蛋白或多肽类分子，基因编辑复合物中的至少一种。
优选地，所述核酸分子为信使RNA(mRNA)、转移RNA(tRNA)、dsRNA、shRNA、DNA、质粒DNA、siRNA、反义寡核苷酸、aiRNA、miRNA中的至少一种；所述基因编辑复合物为mRNA/sgRNA或Cas9/sgRNA。
优选地，所述阳离子脂质类似物和核酸分子的质量比为阳离子脂质类似物：核酸分子＝(2～50)：1；优选地，阳离子脂质类似物：核酸分子＝(5～20)：1。
第五方面，本发明提供一种所述的载药纳米颗粒的制备方法，包括以下步骤：
(1)将所述阳离子脂质类似物的组合物溶于有机溶液，得有机相；
(2)将所述核酸分子溶于缓冲液，得水相；所述水相体积：所述有机相体积＝(2～5)：1；
(3)将所述水相快速加入有机相(或将有机相快速加入所述水相)，混合均匀，透析，即得。
第六方面，本发明将所述的阳离子脂质类似物、所述的阳离子脂质类似物的组合物、所述的载药纳米颗粒在制备、传递或输送分子药物或核酸疫苗中的应用。
与现有技术相比，本发明的有益效果为：
本发明采用醛类化合物、胺类化合物、羧酸类化合物和异腈化合物为原料，通过Ugi反应合成阳离子脂质类似物；本发明阳离子脂质类似物的反应条件温和，合成工艺简单、稳定性好；合成得到的阳离子脂质类似物与固醇、辅助脂质、聚乙二醇脂质衍生物形成脂质纳米颗粒样组合物，该组合物可用于药物输送，包括小分子化合物，mRNA，DNA，siRNA等核酸分子，蛋白/多肽类分子，以及mRNA/sgRNA,Cas9/sgRNA等基因编辑复合物；通过不同给药方式实现在动物体内不同器官的mRNA表达，可满足mRNA核酸疗法、核酸疫苗以及基因编辑等的应用需求。
附图说明
图1为实施例1中A4I18R3C18-2的核磁共振氢谱图。
图2为实施例1中A4I18R3C18-2的质谱图。
图3为实施例2中A1I4R22C18-2的核磁共振氢谱图。
图4为实施例2中A1I4R22C18-2的质谱图。
图5为实施例3中A1I5R22S15的核磁共振氢谱图。
图6为实施例3中A1I5R22S15的质谱图。
图7为实施例4中A1I32R46C18-2的核磁共振氢谱图。
图8为实施例4中A1I32R46C18-2的质谱图。
图9为实施例5中A1I32R47C18-2的核磁共振氢谱图。
图10为实施例5中A1I32R47C18-2的质谱图。
图11为实施例1的化合物制备OVA-mRNA载药纳米颗粒免疫后抗体滴度统计图。
图12为实施例2的化合物制备OVA-mRNA载药纳米颗粒免疫后抗体滴度统计图。
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作进一步说明。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。
实施例中所用的试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1：一种阳离子脂质类似物
以化学反应通式(I)合成阳离子脂质类似物，产物的结构通式为：
Figure PCTCN2022075911-appb-000028
具体制备方法为：
室温下分别将1.0mmol的醛类化合物和1.0mmol的胺类化合物加入到0.5mL甲醇溶液中，室温反应60min后加入1.0mmol羧酸类化合物，室温反应60min后加入1.0mmol异腈化合物，40℃反应24h，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液。
式(I)的合成通式为：
Figure PCTCN2022075911-appb-000029
其中，醛类化合物为A1，A4，A23中任意一种；异腈类化合物为I18；胺类化合物为R1，R2，R3中任意一种；羧酸类化合物为C18，C18-1，C18-2，C18-3，C23中任意一种。
通过上述优选化合物合成的阳离子脂质类似物为如下45种：
A1I18R1C18、A1I18R1C18-1、A1I18R1C18-2、A1I18R1C18-3、A1I18R1C23、A1I18R2C18、A1I18R2C18-1、A1I18R2C18-2、A1I18R2C18-3、A1I18R2C23、A1I18R3C18、A1I18R3C18-1、A1I18R3C18-2、A1I18R3C18-3、A1I18R3C23、A4I18R1C18、A4I18R1C18-1、A4I18R1C18-2、A4I18R1C18-3、A4I18R1C23、A4I18R2C18、A4I18R2C18-1、A4I18R2C18-2、A4I18R2C18-3、A4I18R2C23、A4I18R3C18、A4I18R3C18-1、A4I18R3C18-2、A4I18R3C18-3、A4I18R3C23、A23I18R1C18、A23I18R1C18-1、A23I18R1C18-2、A23I18R1C18-3、A23I18R1C23、A23I18R2C18、A23I18R2C18-1、A23I18R2C18-2、A23I18R2C18-3、A23I18R2C23、A23I18R3C18、A23I18R3C18-1、A23I18R3C18-2、A23I18R3C18-3、A23I18R3C23。
醛类化合物为A1，A4，A23中任意一种；异腈类化合物为I18；胺类化合物为R5，R12中任意一种；羧酸类化合物为C18，C18-2中任意一种。
通过上述优选化合物合成的阳离子脂质类似物为如下12种：
A1I18R5C18、A1I18R5C18-2、A1I18R12C18、A1I18R12C18-2、A4I18R5C18、A4I18R5C18-2、A4I18R12C18、A4I18R12C18-2、A23I18R5C18、A23I18R5C18- 2、A23I18R12C18、A23I18R12C18-2、
醛类化合物为A4或A5；异腈类化合物为I10，I14或I18；胺类化合物为R4，R6，R7，R8，R9，R10或R11；羧酸类化合物为C18或C18-2。
通过上述优选化合物合成的阳离子脂质类似物为如下10种：
A5I18R2C18-2、A4I10R2C18-2、A4I14R2C18-2、A4I18R4C18-2、A4I18R6C18-2、A4I18R7C18-2、A4I18R8C18-2、A4I18R9C18-2、A4I18R10C18-2、A4I18R11C18-2。
其中，A4I18R3C18-2的核磁共振氢谱与质谱图如图1和图2所示。
实施例2：一种阳离子脂质类似物
以化学反应通式(II)合成阳离子脂质类似物，产物的结构通式为：
Figure PCTCN2022075911-appb-000030
具体制备方法为：
室温下分别将1.0mmol的醛类化合物和1.0mmol的胺类化合物加入到0.5mL甲醇溶液中，室温反应60min后加入1.0mmol羧酸类化合物，室温反应60min后加入0.5mmol异腈化合物，40℃反应24h，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液。
式(II)的合成通式为：
Figure PCTCN2022075911-appb-000031
其中，醛类化合物为A1，A4，A21，A23中任意一种；异腈类化合物为I4；胺类化合物为R2，R13，R21，R22，R23中任意一种；羧酸类化合物为C18，C18-1，C18-2中任意一种。
采用上述化合物合成的阳离子脂质类似物为如下60种：
A1I4R2C18、A1I4R2C18-1、A1I4R2C18-2、A1I4R13C18、A1I4R13C18-1、A1I4R13C18-2、A1I4R21C18、A1I4R21C18-1、A1I4R21C18-2、A1I4R22C18、A1I4R22C18-1、A1I4R22C18-2、A1I4R23C18、A1I4R23C18-1、A1I4R23C18-2、A4I4R2C18、A4I4R2C18-1、A4I4R2C18-2、A4I4R13C18、A4I4R13C18-1、A4I4R13C18-2、A4I4R21C18、A4I4R21C18-1、A4I4R21C18-2、A4I4R22C18、A4I4R22C18-1、A4I4R22C18-2、A4I4R23C18、A4I4R23C18-1、A4I4R23C18-2、A21I4R2C18、A21I4R2C18-1、A21I4R2C18-2、A21I4R13C18、A21I4R13C18-1、A21I4R13C18-2、A21I4R21C18、A21I4R21C18-1、A21I4R21C18-2、A21I4R22C18、A21I4R22C18-1、A21I4R22C18-2、A21I4R23C18、A21I4R23C18-1、A21I4R23C18-2、A23I4R2C18、A23I4R2C18-1、A23I4R2C18-2、A23I4R13C18、A23I4R13C18-1、A23I4R13C18-2、A23I4R21C18、A23I4R21C18-1、A23I4R21C18-2、A23I4R22C18、A23I4R22C18-1、A23I4R22C18-2、A23I4R23C18、A23I4R23C18-1、A23I4R23C18-2。
醛类化合物为A1；异腈类化合物为I4；胺类化合物为R27，R28，R29，R30，R31，R32，R33，R34，R35，R36，R42，R43，R48中任意一种；羧酸类化合物为C18-2。
采用上述化合物合成的阳离子脂质类似物为如下13种：
A1I4R27C18-2、A1I4R28C18-2、A1I4R29C18-2、A1I4R30C18-2、A1I4R31C18-2、A1I4R32C18-2、A1I4R33C18-2、A1I4R34C18-2、A1I4R35C18-2、A1I4R36C18-2、A1I4R42C18-2、A1I4R43C18-2、A1I4R48C18-2。
醛类化合物为A1中任意一种；异腈类化合物为I4；胺类化合物为R22；羧酸类化合物为C12，C14，C16，S11，S13，S15，S17，S19中任意一种。
采用上述化合物合成的阳离子脂质类似物为如下8种：
A1I4R22C12、A1I4R22C14、A1I4R22C16、A1I4R22S11、A1I4R22S13、A1I4R22S15、A1I4R22S17、A1I4R22S19。
其中，A1I4R22C18-2的核磁共振氢谱与质谱图如图3和图4所示。
实施例3：一种阳离子脂质类似物
以化学反应通式(III)合成阳离子脂质类似物，产物的结构通式为：
Figure PCTCN2022075911-appb-000032
具体制备方法为：
室温下分别将1.0mmol的醛类化合物和1.0mmol的胺类化合物加入到0.5mL甲醇溶液中，室温反应60min后加入1.0mmol羧酸类化合物，室温反应60min后加入0.33mmol异腈化合物，40℃反应24h，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液。
式(III)的合成通式为：
Figure PCTCN2022075911-appb-000033
其中，醛类化合物为A1；异腈化合物为I5；胺类化合物为R2，R13，R21，R22，R23，R26，R27，R28，R29，R30，R31，R32，R33，R34，R35，R36，R37，R38，R39，R40，R41或R50；羧酸类化合物为C8，C10，C12，C14，C16，C18，C18-2，C21，S11，S13，S15，S17或S19。
采用上述化合物合成的阳离子脂质类似物为如下29种：
A1I5R22C8、A1I5R22C10、A1I5R22C12、A1I5R22C14、A1I5R22C16、A1I5R22C18、A1I5R22C18-2、A1I5R22S11、A1I5R22S13、A1I5R22S15、A1I5R22S17、A1I5R22S19、A1I5R26S15、A1I5R27S15、A1I5R28S15、A1I5R29S15、A1I5R30S15、A1I5R31S15、A1I5R32S15、A1I5R33S15、A1I5R34S15、A1I5R35S15、A1I5R36S15、A1I5R37S15、A1I5R38S15、A1I5R39S15、A1I5R40S15、A1I5R41S15、A1I5R50C21。
其中，A1I5R22S15的核磁共振氢谱与质谱图如图5和图6所示。
实施例4：一种阳离子脂质类似物
以化学反应通式(IV)合成阳离子脂质类似物，产物的结构通式为：
Figure PCTCN2022075911-appb-000034
具体制备方法为：
室温下分别将1.0mmol的醛类化合物和0.5mmol的胺类化合物加入到0.5mL甲醇溶液中，室温反应60min后加入1.0mmol羧酸类化合物，室温反应60min后加入1.0mmol异腈化合物，40℃反应24h，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液。
式(IV)的合成通式为：
Figure PCTCN2022075911-appb-000035
其中，醛类化合物为A1；异腈类化合物为I10、I12、I31或I32；胺类化合物为R46或R51；羧酸类化合物为C8或C18-2。
采用上述化合物合成的阳离子脂质类似物为如下4种：
A1I12R51C8、A1I10R51C18-2、A1I31R46C18-2、A1I32R46C18-2。
其中，A1I32R46C18-2的核磁共振氢谱与质谱图如图7和图8所示。
实施例5：一种阳离子脂质类似物
以化学反应通式(V)合成阳离子脂质类似物，产物的结构通式为：
Figure PCTCN2022075911-appb-000036
具体制备方法为：
室温下分别将1.0mmol的醛类化合物和0.33mmol的胺类化合物加入到0.5mL甲醇溶液中，室温反应60min后加入1.0mmol羧酸类化合物，室温反应60min后加入1.0mmol异腈化合物，40℃反应24h，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液。
式(V)的合成通式为：
Figure PCTCN2022075911-appb-000037
其中，醛类化合物为A1；异腈类化合物为I12、I31或I32；胺类化合物为R47；羧酸类化合物为C21或C18-2。
采用上述化合物合成的阳离子脂质类似物为如下3种：
A1I12R47C21、A1I31R47C18-2、A1I32R47C18-2。
其中，A1I32R47C18-2的核磁共振氢谱与质谱图如图9和图10所示。
实施例6：一种载药纳米颗粒
该载药纳米颗粒包括阳离子脂质类似物、胆固醇、二硬脂酰磷脂酰胆碱(DSPC)、1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)。
其中，采用摩尔比计，阳离子脂质类似物：胆固醇：DSPC：DMG-PEG＝50：38.5：10：1.5。
该载药米粒颗粒中，阳离子脂质类似物和mRNA或siRNA等核酸药物质量比为阳离子脂质类似物：核酸药物＝11：1。
以负载mRNA的该载药纳米颗粒的制备方法为例：
将阳离子脂质类似物、胆固醇、DSPC、DMG-PEG溶于乙醇溶液，按上述摩尔比混合得有机相；将对应质量比的mRNA核酸药物溶于醋酸钠缓冲溶液(25mM，pH＝3.0)得水相，使水相体积：有机相体积＝3：1；将水相快速加入有机相中混合均匀，静置10分钟后装入1000Da透析袋中，在4℃1×PBS溶液中透析2小时，即得到可直接用于注射的纳米颗粒。
表1使用实施例6制备脂质纳米颗粒表征数据
实施例 阳离子脂质类似物名称 粒径(nm) PDI Zeta电位(mV) 包封率
1 A4I18R3C18-2 83±9 0.12 -3.2 75.3％
2 A1I4R22C18-2 133±6 0.17 -5.5 62.1％
3 A1I5R22S15 144±7 0.22 -7.2 65.4％
4 A1I32R46C18-2 110±11 0.20 -2.5 67.2％
5 A1I32R47C18-2 150±9 0.21 -3.1 60.5％
实施例7：一种载药纳米颗粒
该载药纳米颗粒包括阳离子脂质类似物、β-谷固醇、二油酰磷脂酰乙醇胺 (DOPE)、1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)。
其中，采用摩尔比计，阳离子脂质类似物：β-谷固醇：DOPE：DMG-PEG＝35：25：5：2。该载药纳米颗粒中，阳离子脂质类似物和mRNA或siRNA等核酸药物质量比为阳离子脂质类似物：核酸药物＝5：1。
实施例8：一种载药纳米颗粒
该载药纳米颗粒包括阳离子脂质类似物、豆甾醇、神经酰胺磷酸胆碱、1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)。
其中，采用摩尔比计，阳离子脂质类似物：豆甾醇：神经酰胺磷酸胆碱：DMG-PEG＝55：40：12：2.5。
该载药米粒颗粒中，阳离子脂质类似物和mRNA或siRNA等核酸药物质量比为阳离子脂质类似物：核酸药物＝15：1。
实施例9：一种载药纳米颗粒
该载药纳米颗粒包括阳离子脂质类似物、胆固醇琥珀酸单酯(胆固醇衍生物)、鞘磷脂、1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)。
其中，采用摩尔比计，阳离子脂质类似物：胆固醇琥珀酸单酯：鞘磷脂：DMG-PEG＝60：45：15：3.5。
该载药纳米颗粒中，阳离子脂质类似物和mRNA或siRNA等核酸药物质量比为阳离子脂质类似物：核酸药物＝18：1。
实施例10：动物实验检测静脉注射mRNA体内递送表达水平
以Luciferase-mRNA作为模型mRNA；以实施例1～5的阳离子脂质类似物按照实施例6的方法制备载药纳米颗粒。
具体操作方法如下：
向6～8周龄的C57BL/6小鼠尾静脉注射200μL含5μg Luciferase-mRNA的纳米颗粒或上述制备的载药纳米颗粒，6小时后向小鼠腹腔注射200μL含3mg D-Luciferin potassium salt的1×PBS溶液，等待10min后通过生物发光成像来评价体内mRNA表达情况。
载药纳米颗粒的体内表达情况如表2至表6所示：
表2实施例1的化合物制备mRNA载药纳米颗粒静脉注射的体内表达情况
Figure PCTCN2022075911-appb-000038
Figure PCTCN2022075911-appb-000039
Figure PCTCN2022075911-appb-000040
Figure PCTCN2022075911-appb-000041
Figure PCTCN2022075911-appb-000042
Figure PCTCN2022075911-appb-000043
Figure PCTCN2022075911-appb-000044
表3实施例2的化合物制备mRNA载药纳米颗粒静脉注射的体内表达情况
Figure PCTCN2022075911-appb-000045
Figure PCTCN2022075911-appb-000046
Figure PCTCN2022075911-appb-000047
Figure PCTCN2022075911-appb-000048
Figure PCTCN2022075911-appb-000049
Figure PCTCN2022075911-appb-000050
表4实施例3的化合物制备mRNA载药纳米颗粒静脉注射的体内表达情况
Figure PCTCN2022075911-appb-000051
Figure PCTCN2022075911-appb-000052
Figure PCTCN2022075911-appb-000053
Figure PCTCN2022075911-appb-000054
Figure PCTCN2022075911-appb-000055
Figure PCTCN2022075911-appb-000056
Figure PCTCN2022075911-appb-000057
Figure PCTCN2022075911-appb-000058
Figure PCTCN2022075911-appb-000059
Figure PCTCN2022075911-appb-000060
Figure PCTCN2022075911-appb-000061
表5实施例4的化合物制备mRNA载药纳米颗粒静脉注射的体内表达情况
Figure PCTCN2022075911-appb-000062
表6实施例5的化合物制备mRNA载药纳米颗粒静脉注射的体内表达情况
Figure PCTCN2022075911-appb-000063
Figure PCTCN2022075911-appb-000064
实施例11：动物实验检测肌肉注射mRNA体内递送表达水平
以Luciferase-mRNA作为模型mRNA；以实施例1～5的阳离子脂质类似物 按照实施例6的方法制备载药纳米颗粒。
具体操作方法如下：
向6～8周龄的C57BL/6小鼠腿部肌肉注射100μL含5μg Luciferase-mRNA的纳米颗粒或上述制备的载药纳米颗粒，6小时后向小鼠腹腔注射200μL含3mg D-Luciferin potassium salt的1×PBS溶液，等待10min后通过生物发光成像来评价体内mRNA表达情况。
表7实施例1的化合物制备mRNA载药纳米颗粒肌肉注射的体内表达情况
Figure PCTCN2022075911-appb-000065
Figure PCTCN2022075911-appb-000066
Figure PCTCN2022075911-appb-000067
表8实施例2的化合物制备mRNA载药纳米颗粒肌肉注射的体内表达情况
Figure PCTCN2022075911-appb-000068
Figure PCTCN2022075911-appb-000069
Figure PCTCN2022075911-appb-000070
Figure PCTCN2022075911-appb-000071
表9实施例3的化合物制备mRNA载药纳米颗粒肌肉注射的体内表达情况
Figure PCTCN2022075911-appb-000072
Figure PCTCN2022075911-appb-000073
Figure PCTCN2022075911-appb-000074
Figure PCTCN2022075911-appb-000075
Figure PCTCN2022075911-appb-000076
Figure PCTCN2022075911-appb-000077
Figure PCTCN2022075911-appb-000078
表10实施例4的化合物制备mRNA载药纳米颗粒肌肉注射的体内表达情况
Figure PCTCN2022075911-appb-000079
Figure PCTCN2022075911-appb-000080
表11实施例5的化合物制备mRNA载药纳米颗粒肌肉注射的体内表达情况
Figure PCTCN2022075911-appb-000081
Figure PCTCN2022075911-appb-000082
实施例12：动物实验检测腹腔注射mRNA体内递送表达水平
以Luciferase-mRNA作为模型mRNA；以实施例2的阳离子脂质类似物A1I4R22C18-2按照实施例6的方法制备载药纳米颗粒。
具体操作方法如下：
向6～8周龄的C57BL/6小鼠腹腔注射200μL含5μg Luciferase-mRNA的纳米颗粒或上述制备的载药纳米颗粒，6小时后向小鼠腹腔注射200μL含3mg D-Luciferin potassium salt的1×PBS溶液，等待10min后通过生物发光成像来评价体内mRNA表达情况。
表12实施例2的化合物制备mRNA载药纳米颗粒腹腔注射的体内表达情况
Figure PCTCN2022075911-appb-000083
实施例13：动物实验检测气管注射mRNA体内递送表达水平
以Luciferase-mRNA作为模型mRNA；以实施例2的阳离子脂质类似物A1I4R22C18-2按照实施例6的方法制备载药纳米颗粒。
具体操作方法如下：
向6～8周龄的C57BL/6小鼠气管注射100μL含5μg Luciferase-mRNA的纳米颗粒或上述制备的载药纳米颗粒，6小时后向小鼠腹腔注射200μL含3mg D-Luciferin potassium salt的1×PBS溶液，等待10min后通过生物发光成像来评价体内mRNA表达情况。
表13实施例2的化合物制备mRNA载药纳米颗粒气管注射的体内表达情况
Figure PCTCN2022075911-appb-000084
实施例14：动物实验评估体内递送mRNA的免疫性能
以OVA-mRNA作为模型mRNA；以实施例1和2的阳离子脂质类似物按照实施例6的方法制备载药纳米颗粒。
具体操作方法如下：
选6～8周龄的C57BL/6小鼠于SPF级动物房实验，每组选取5只小鼠，向每只小鼠腿部肌肉或腹腔注射100μL含5μg OVA-mRNA的纳米颗粒或上述制备的载药纳米颗粒，使用相同剂量再加强一次后，取血通过酶联免疫吸附测定(ELISA)用于抗体滴度分析。
以实施例1中阳离子脂质类似物为例，在第0，14天分别肌肉注射5μg OVA-mRNA的纳米颗粒或上述制备的载药纳米颗粒后，在第21天取血进行抗体滴度 分析，结果如图11所示。
以实施例2中阳离子脂质类似物A1I4R22C18-2为例，在第0，7天分别肌肉或腹腔注射5μg OVA-mRNA的纳米颗粒或上述制备的载药纳米颗粒后，在第14天取血进行抗体滴度分析，结果如图12所示。
实施例15：动物实验检测静脉注射siRNA体内递送水平
以Cy5-siRNA作为模型siRNA；以实施例1的阳离子脂质类似物A4I18R2C18-2和A1I4R22C18-2按照实施例6的方法制备载药纳米颗粒。
具体操作方法如下：
向6～8周龄的C57BL/6小鼠腿部静脉注射200μL含10μg Cy5-siRNA的纳米颗粒或上述制备的载药纳米颗粒，1小时后通过生物荧光成像来评价体内siRNA递送情况。
表14实施例1的化合物制备siRNA载药纳米颗粒静脉注射的体内递送情况
Figure PCTCN2022075911-appb-000085
表15实施例2的化合物制备siRNA载药纳米颗粒静脉注射的体内递送情况
Figure PCTCN2022075911-appb-000086
Figure PCTCN2022075911-appb-000087
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 (21)

  1. 一种阳离子脂质类似物，其特征在于，其具有如式(I)～式(Ⅴ)任一种所示结构：
    Figure PCTCN2022075911-appb-100001
    式中，m 1为独立地选自氢原子、直链烷基、支链烷基、直链烯基、支链烯基、取代炔基、环烷基、苯基、含杂原子芳香基中的至少一种基团；
    m 2为独立地选自直链烷基、直链烯基、含杂原子直链烷基、含杂原子直链 亚烷基、含杂原子支链烷基、含杂原子支链亚烷基、含杂原子环的烷基、含杂原子环的亚烷基、含酯基的烷基、含醚基的烷基中的至少一种基团；
    m 3为独立地选自直链烷基、直链烯基、环烷基、含杂原子直链烷基、含杂原子支链烷基、含杂原子支链亚烷基、含杂原子环的烷基、含杂原子和芳香环的烷基、含羟基的烷基、含酯基的烷基、含醚基的烷基、含磺酸酯基的烷基中的至少一种基团；
    m 4为独立地选自直链烷基、支链烷基、直链烯基、支链烯基、直链炔基、含杂原子直链烷基、含杂原子支链烷基、含杂原子环的烷基中的至少一种基团。
  2. 根据权利要求1所述的阳离子脂质类似物，其特征在于，式中，m 1为独立地选自
    Figure PCTCN2022075911-appb-100002
    Figure PCTCN2022075911-appb-100003
    和/或
    m 2为独立地选自
    Figure PCTCN2022075911-appb-100004
    Figure PCTCN2022075911-appb-100005
    Figure PCTCN2022075911-appb-100006
    和/或
    m 3为独立地选自
    Figure PCTCN2022075911-appb-100007
    Figure PCTCN2022075911-appb-100008
    Figure PCTCN2022075911-appb-100009
    和/或
    m 4为独立地选自
    Figure PCTCN2022075911-appb-100010
    Figure PCTCN2022075911-appb-100011
    Figure PCTCN2022075911-appb-100012
  3. 根据权利要求2所述的阳离子脂质类似物，其特征在于，所述m 1为
    Figure PCTCN2022075911-appb-100013
    Figure PCTCN2022075911-appb-100014
    和/或
    所述m 2为、
    Figure PCTCN2022075911-appb-100015
    Figure PCTCN2022075911-appb-100016
    Figure PCTCN2022075911-appb-100017
    和/或
    所述m 3为
    Figure PCTCN2022075911-appb-100018
    Figure PCTCN2022075911-appb-100019
    Figure PCTCN2022075911-appb-100020
    和/或
    所述m 4为
    Figure PCTCN2022075911-appb-100021
    Figure PCTCN2022075911-appb-100022
  4. 一种权利要求1-3任一项权利要求所述的阳离子脂质类似物的制备方法，其特征在于，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腈化合物，再于4～60℃反应6～72h，分离提纯产物，即成。
  5. 根据权利要求4所述的阳离子脂质类似物的制备方法，其特征在于，所述醛类化合物、胺类化合物、羧酸类化合物和异腈化合物的摩尔比为(0.1～1.5)：(0.1～1.5)：(0.1～1.5)：(0.1～1.5)。
  6. 根据权利要求4所述的阳离子脂质类似物的制备方法，其特征在于，所述异腈化合物为单官能团异腈化合物、双官能团异腈化合物、三官能团异腈化合物中的一种。
  7. 根据权利要求5所述的阳离子脂质类似物的制备方法，其特征在于，所述异腈化合物为如下化合物I3～I38中的任一种：
    Figure PCTCN2022075911-appb-100023
  8. 根据权利要求4所述的阳离子脂质类似物的制备方法，其特征在于，所述醛类化合物为单官能团醛类化合物中的一种。
  9. 根据权利要求8所述的阳离子脂质类似物的制备方法，其特征在于，所述醛类化合物为如下化合物A1～A35中的任一种：
    Figure PCTCN2022075911-appb-100024
  10. 根据权利要求4所述的阳离子脂质类似物的制备方法，其特征在于，所述胺类化合物为单官能团胺类化合物、双官能团胺类化合物、三官能团胺类化合物中的一种。
  11. 根据权利要求10所述的阳离子脂质类似物的制备方法，其特征在于，优选地所述胺类化合物为如下化合物R1～R53中的任一种：
    Figure PCTCN2022075911-appb-100025
  12. 根据权利要求4所述的阳离子脂质类似物的制备方法，其特征在于，羧酸类化合物为如下化合物C8～C30或S11～S19中的任一种：
    Figure PCTCN2022075911-appb-100026
  13. 一种权利要求1-3任一项权利要求所述的阳离子脂质类似物的组合物，其特征在于，包括所述阳离子脂质类似物和固醇、辅助脂质、聚乙二醇脂质衍生物中的至少一种。
  14. 根据权利要求13所述阳离子脂质类似物的组合物，其特征在于，所述固醇包括胆固醇、谷固醇、豆甾醇、胆固醇衍生物中的至少一种；所述辅助脂质为中性磷脂。
  15. 根据权利要求13所述阳离子脂质类似物的组合物，其特征在于，所述阳离子脂质类似物、胆固醇、辅助脂质、聚乙二醇脂质衍生物的摩尔比为(20～70)：(20～50)：(2～30)：(0.1～20)。
  16. 一种载药纳米颗粒，其特征在于，包括权利要求13所述的阳离子脂质类似物的组合物和药物。
  17. 根据权利要求16所述的载药纳米颗粒，其特征在于，所述药物包括小 分子化合物，核酸分子，蛋白或多肽类分子，基因编辑复合物中的至少一种。
  18. 根据权利要求17所述的载药纳米颗粒，其特征在于，所述核酸分子为信使RNA、转移RNA、dsRNA、shRNA、DNA、质粒DNA、siRNA、反义寡核苷酸、aiRNA、miRNA中的至少一种；所述基因编辑复合物为mRNA/sgRNA或Cas9/sgRNA。
  19. 根据权利要求17所述的载药纳米颗粒，其特征在于，所述阳离子脂质类似物和核酸分子的质量比为阳离子脂质类似物：核酸分子＝(2～50)：1。
  20. 一种权利要求19所述的载药纳米颗粒的制备方法，其特征在于，包括以下步骤：
    (1)将所述阳离子脂质类似物的组合物溶于有机溶液，得有机相；
    (2)将所述核酸分子溶于缓冲液，得水相；
    所述水相体积：所述有机相体积＝(2～5)：1；
    (3)将所述水相与所述有机相快速混合均匀，透析，即得。
  21. 权利要求1-3任一项权利要求所述的阳离子脂质类似物、权利要求13-15任一项权利要求所述的阳离子脂质类似物的组合物、权利要求16-19任一项权利要求所述的载药纳米颗粒在制备、传递或输送分子药物或核酸疫苗中的应用。
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