WO2023109243A1 - 可离子化脂质化合物及核酸体外细胞转染试剂 - Google Patents

可离子化脂质化合物及核酸体外细胞转染试剂 Download PDF

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WO2023109243A1
WO2023109243A1 PCT/CN2022/121381 CN2022121381W WO2023109243A1 WO 2023109243 A1 WO2023109243 A1 WO 2023109243A1 CN 2022121381 W CN2022121381 W CN 2022121381W WO 2023109243 A1 WO2023109243 A1 WO 2023109243A1
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integer
nucleic acid
lipid
ionizable lipid
lipid compound
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French (fr)
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崔艳芳
刘滨磊
吉帅洁
张宝倩
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武汉滨会生物科技股份有限公司
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the disclosure belongs to the technical field of in vitro cell transfection reagents, in particular to ionizable lipid compounds and nucleic acid transfection reagents.
  • Cell transfection refers to the technique of introducing exogenous molecules such as DNA, RNA, etc. into eukaryotic cells. With the continuous development of molecular biology and cell biology research, transfection has become a routine tool to study and control gene function in eukaryotic cells. It is used more and more widely in biological experiments such as studying gene function, regulating gene expression, mutation analysis and protein production.
  • DNA cell transfection methods are mainly divided into three categories: physical mediation (electroporation, gene gun method, microinjection method), chemical mediation (lipofection method, calcium phosphate coprecipitation method, Cationic polymer-mediated method), biological mediation (virus-mediated transfection, protoplast transfection).
  • the disadvantage of electroporation is high cell lethality; the disadvantage of microinjection and gene gun is that the operation is complicated, the required equipment is expensive, and it will cause the deletion or mutation of nucleic acid fragments; the disadvantage of protoplast transfection is low transfection efficiency and high cost
  • the disadvantage of the calcium phosphate co-precipitation method is that it requires high DNA concentration and has limited applicability; the cationic liposome transfection method has certain requirements for DNA concentration and has certain toxicity to cells.
  • Virus-mediated transfection technology although the transfection efficiency is high and has the advantage of low cytotoxicity, however, the preparation procedure of the virus transfection method is complicated and often has strong selectivity for cell types. Difficult to popularize.
  • cationic liposomes which are positively charged liposomes, which form DNA-liposome complexes by electrostatic interaction and DNA binding, and then endocytosis by cells It has the advantages of simple operation and wide applicability, but the transfection efficiency of different cells needs to be further improved, and there is still a certain degree of toxicity to cells.
  • An ideal cell transfection reagent should have the advantages of high transfection efficiency and low cytotoxicity.
  • the existing cationic liposome transfection reagents have obvious advantages compared with viral vectors and other types of non-viral vectors in terms of encapsulation efficiency, transfection efficiency and cytotoxicity, there is still certain cytotoxicity, and the transfection Efficiency also needs to be further improved. Therefore, more in vitro cell transfection reagents with high transfection efficiency and low cytotoxicity need to be developed to provide more options for cell transfection.
  • the purpose of this disclosure is to provide an ionizable lipid compound that is easy to combine with DNA and RNA and is easy to degrade, which enriches the types of ionizable lipid compounds and can be used to prepare DNA in vitro cell transfection reagents or RNA in vitro cell transfection reagents.
  • Transfection reagents provide more options for DNA transfection or RNA transfection in vitro.
  • Another object of the present disclosure is to provide a nucleic acid transfection reagent in vitro with high transfection efficiency and low cytotoxicity, which can be used for DNA transfection or RNA transfection in vitro.
  • An ionizable lipid compound is one or more of the compounds shown in general formula (I), general formula (II), and general formula (III):
  • R 1 is hydrogen, methyl, ethyl or isopropyl.
  • n is an integer between 1 and 10, for example, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • n is an integer between 1 and 3, for example n is 1, 2 or 3.
  • f is an integer between 1 and 5, for example f is 1, 2, 3, 4 or 5.
  • x is an integer between 1 and 8, for example x is 1, 2, 3, 4, 5, 6, 7 or 8.
  • y is an integer between 1 and 9, for example, y is 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • R2 and R3 are independently hydrogen, methyl, ethyl or isopropyl.
  • p is an integer between 1 and 5, for example, p is 1, 2, 3, 4 or 5.
  • q is an integer between 1 and 3, for example, q is 1, 2 or 3.
  • said R 1 is hydrogen
  • one of said R2 and said R3 is hydrogen, and the other is methyl, ethyl or isopropyl.
  • one of said R2 and said R3 is hydrogen, and the other is methyl.
  • the m is an integer between 3-8. In some other embodiments, m is an integer between 4-6.
  • the f is an integer between 1-4. In other embodiments, f is 2 or 3.
  • the x is an integer between 2-5. In some other embodiments, x is an integer between 2-4.
  • the y is an integer between 3-9. In some other embodiments, y is an integer between 5-9.
  • R 1 is hydrogen, m is an integer between 4 and 6, n is 2, and f is an integer between 1 and 3.
  • x is 3, and y is an integer between 6-9.
  • R 2 is methyl
  • R 3 is hydrogen
  • p is 5
  • q is an integer between 1 and 3.
  • the ionizable lipid compound is one or more of the compounds shown in the following structural formula:
  • a nucleic acid in vitro cell transfection reagent comprising the composition of the ionizable lipid compound and the formation of auxiliary lipid molecules according to any one of claims 1 to 7.
  • the nucleic acid includes deoxyribonucleic acid and ribonucleic acid, the deoxyribonucleic acid is plasmid DNA, and the ribonucleic acid is mRNA.
  • the auxiliary lipid molecules include cholesterol, calcipotriol, stigmasterol, ⁇ -sitosterol, betulin, ursolic acid, lupeol, oleanolic acid, dioleoyl phospholipids Acylcholine, Distearoylphosphatidylcholine, 1-Stearyl-2-Oleoyl Lecithin, Dioleoyl Phosphatidylethanolamine, (1,2-Dioleoxypropyl) Trimethyl Chloride Ammonium, Didecyldimethylammonium Bromide, 1,2-Dimyristoyl-sn-Glycero-3-Ethylphosphocholine, Dipalmitoylphosphatidylethanolamine-Methoxypolyethylene Glycol 5000, One or more of one or more of distearoylphosphatidylethanolamine-macrogol 2000, activated carbon, silicon dioxide, and calcium phosphate.
  • the molar ratio of the ionizable lipid compound to the auxiliary lipid molecule is 1:(0.5-2).
  • the mass ratio of the nucleic acid to the composition is 1:(5-50). It is further preferably 1:(5-40) in some embodiments. In some embodiments it is 1:(5-30). In some embodiments it is 1:(5-20).
  • the N:P ratio of the nucleic acid to the composition is 5-10:1.
  • the N:P ratio of the nucleic acid to the composition is 5-8:1.
  • Ionizable lipid molecules and their composition have recently been developed into important drug delivery carriers, but there are still few examples of transfection of cells in vitro for DNA, and no reports for transfection of cells in vitro for RNA.
  • the composition of ionizable lipid molecules has obvious advantages of less toxicity as transfection reagents for DNA in vitro cells, and at the same time, the composition of a series of ionizable lipid compounds we invented is suitable for the suspension of difficult-to-transfect DNA
  • the transfection efficiency of cells such as Jurkat cells is significantly higher than lipo2000, another commonly used lipofection reagent.
  • the application of the ionizable lipid compound and its composition in the fields of DNA in vitro cell transfection and mRNA in vitro cell transfection will promote the research and wide application of eukaryotic cell gene function.
  • the embodiments of the present disclosure provide a new ionizable lipid compound, which enriches the types of ionizable lipid compounds, and provides more options for deoxyribonucleic acid and ribonucleic acid transfection in vitro cells, and the formed liposomes
  • the particles have uniform size, good stability, high encapsulation efficiency, high delivery efficiency and low toxicity. They can be used as transfection reagents for in vitro cell DNA transfection and RNA transfection, which is of great significance to the development and application of related fields.
  • Fig. 1 is the hydrogen spectrogram of compound 1-1
  • Fig. 2 is the hydrogen spectrogram of 2-(bis(2-aminoethyl)amino)ethan-1-alcohol);
  • Fig. 3 is the hydrogen spectrogram of compound 1
  • Fig. 4 is the mass spectrogram of compound 1;
  • Fig. 5 is the hydrogen spectrogram of compound 2-1;
  • Fig. 6 is the hydrogen spectrogram of compound 2
  • Fig. 7 is the mass spectrogram of compound 2;
  • Fig. 8 is the hydrogen spectrogram of compound 3-1
  • Fig. 9 is the hydrogen spectrogram of 1,3-diamino-2-propanol
  • Figure 10 is the hydrogen spectrogram of compound 3
  • Fig. 11 is the mass spectrogram of compound 3.
  • Figure 12 is a transmission electron microscope image and a zeta potential test result graph of pDNA lipid nanoparticle Lipid-1;
  • Fig. 13 is the particle size distribution figure of mRNA lipid nanoparticle
  • Fig. 14 is the gel retardation experiment electrophoresis figure of mRNA lipid nanoparticle Lipid-3;
  • Fig. 15 is the gel retardation experiment electrophoresis figure of the pDNA lipid nanoparticles under different pDNA and lipid molecular dosage ratios;
  • Figure 16 is the change over time of the average fluorescence intensity of mRNA lipid nanoparticles Lipid-1 transfected 293T cells and Hela cells;
  • Figure 17 is the in vitro transfection fluorescence micrograph and the proportion of positive cells of mRNA lipid nanoparticle Lipid-1 to the suspension cell line Jurkat cells;
  • Fig. 18 is the fluorescence micrograph of in vitro transfection of pDNA lipid nanoparticles Lipid-1 to various adherent cells (MCF-7 cells, Hela cells and 293T cells);
  • Figure 19 is a graph showing the experimental results of the stability test of mRNA nano-lipid particles in the environment of RNA hydrolase
  • Fig. 20 is a graph showing the results of toxicity assay of lipid nanoparticle Lipid-1 transfected into 293T cells in vitro.
  • Ionizable lipid molecules are usually divided into three parts, including head group, linker group and tail group.
  • the head group (Head) can be protonated in a weakly acidic environment, so that the ionizable lipids are positively charged as a whole, and the ionizable lipid molecules combine with the negatively charged nucleic acids to form a compact spherical structure, that is, the formation of Nano-lipid particles, after entering the cell, the nano-lipid particles first enter the early endosome, then enter the late endosome, and finally enter the lysosome, where most of the nucleic acids will be degraded by enzymes in the endoplasmic reticulum.
  • the acidity of late endosomes decreases, which will promote the protonation of lipid nanoparticles and destroy the endosomal membrane. Some lipid nanoparticles can escape through endosomes to protect nucleic acids from degradation, thus avoiding premature degradation of nucleic acids.
  • Linker is another important functional part, which is used to connect the head group and tail group of ionizable lipid molecules. This site will not only affect the overall pKa of the liposome, but also affect the potential and biodegradability of the liposome.
  • the tail group (Tail) is the third most important functional part, which usually has a long hydrophobic carbon chain and makes the long lipid chain have "lipid properties". Therefore, the structures of the head group, the linker group and the tail group are all related to the transfection efficiency and toxicity degree of liposomes.
  • the inventors have carried out sufficient research from the three parts of the head group, the linker group and the tail group, and finally provided the ionizable lipid compound shown in the general formula (I), the general formula (II) or the general formula (III),
  • R is hydrogen, methyl, ethyl or isopropyl
  • n 1 and 10;
  • n is an integer between 1 and 3;
  • f is an integer between 1 and 5;
  • x is an integer between 1 and 8;
  • y is an integer between 1 and 9;
  • R2 and R3 are independently hydrogen, methyl, ethyl or isopropyl
  • p is an integer between 1 and 5;
  • q is an integer between 1 and 3.
  • the multiple tertiary amine groups and the special symmetrical head group structure in the ionizable lipid compound of the disclosed embodiment are protonated and positively charged in an acidic environment, which improves the binding ability to negatively charged nucleic acids , and prevent premature degradation of nucleic acids in cells by nucleolytic enzymes.
  • R 1 is hydrogen, m is an integer between 1 and 10, n is an integer between 1 and 3, and f is an integer between 1 and 5; in another In some embodiments, R is hydrogen, m is an integer between 3 and 8, n is an integer between 1 and 3, and f is an integer between 1 and 4; in other embodiments, R is Hydrogen, m is an integer between 4 and 6, n is an integer between 1 and 3, f is an integer between 1 and 3; in other embodiments, R1 is hydrogen, m is between 4 and 6 An integer between, n is 2, and f is an integer between 1 and 3.
  • x is an integer between 1 and 6, and y is an integer between 3 and 9; in other embodiments, x is an integer between 2 and 5, y is an integer between 5 and 9; in other embodiments, x is an integer between 2 and 4, and y is an integer between 6 and 9; in other embodiments, x is 3, and y is An integer between 6 and 9.
  • R 2 and R 3 are independently hydrogen or methyl, p is an integer between 1 and 5, and q is an integer between 1 and 3; in other implementations
  • R2 is methyl
  • R3 is hydrogen
  • p is an integer between 1 and 5
  • q is an integer between 1 and 3
  • R2 is methyl
  • R3 is hydrogen
  • p is an integer between 2 and 5
  • q is an integer between 1 and 3
  • R2 is methyl
  • R3 is hydrogen
  • p is 5, and q is between 1 and 3 Integer between.
  • a suitable tail group structure can improve the efficiency of the nucleic acid delivery system.
  • the ionizable lipid compound of the embodiments of the present disclosure has multiple identical hydrocarbon tails, which is beneficial for the nucleic acid-loaded liposome to pass through the cell membrane, and is applicable to various types of cells
  • the efficiency of nucleic acid delivery is equivalent to or higher than that of existing commercialized transfection reagents. Combined with the special structural design of its linker, it can achieve effective degradation, thereby achieving rapid intracellular clearance and reducing the toxic and side effects of liposomes. .
  • the liposome formed by the ionizable lipid compound of the embodiment of the present disclosure is positively charged in the acidic environment of the late endosome, so that it can interact with the phospholipid anion in the endosome membrane to form a liposome adapting to the non-bilayer structure. Ion pairs, and then destroy the endosomal membrane, and realize the escape of endosomes to break through the barriers in the cell. Thus, nucleic acids can be efficiently delivered into the cytoplasm, with subsequent efficient gene expression.
  • the linker of the ionizable lipid compound of the disclosed embodiment is a designed ester group, which makes the liposome easy to degrade and reduces cytotoxicity.
  • the liposomes formed by the ionizable lipid compounds of the embodiments of the present disclosure show good transfection efficiency in various types of cells, indicating that it is effective and effective as a general nucleic acid transfection reagent application. Uniqueness of transfectable suspension cells.
  • Lipid-1 only refers to the use of Compound 1 prepared in Example 1 as a nano-lipid particle formed by an ionizable lipid compound and other components
  • Lipid-2 only refers to Compound 2 prepared in Example 2 As a nano lipid particle formed by an ionizable lipid compound and other components
  • Lipid-3 only refers to the use of compound 3 prepared in Example 3 as a nano lipid particle formed by an ionizable lipid compound and other components, for each
  • the type of nucleic acid encapsulated in the nanolipid particle is not limited.
  • Embodiment 4 the preparation of nano-lipid particle, the mensuration of particle size and electric potential:
  • pDNA nano-lipid particles which are respectively labeled as Lipid-1, Lipid-2 and Lipid-2.
  • Lipid-3 qualified lipid nanoparticles can be used to transfect prepared cells. Utilize Zetasizer Nano ZS (Malvern, Worcestershire, UK) to detect the particle size of nano liposome particles, PDI.
  • the measurement of particle diameter is measured after diluting nano liposome particle solution 50 times with 1 * PBS, and the mensuration of Zeta potential is to measure by nano liposome particle diluted in the PBS of 15mM, and test result is as shown in Table 1 and Fig. 12 shown.
  • Table 1 and Figure 12 show that the average particle size of pDNA nano-lipid particles Lipid-1, Lipid-2 and Lipid-3 is in the range of 120-150nm, the particle size distribution is uniform, the encapsulation efficiency is above 90%, and the potential is at -18.6 ⁇ -11.6mV.
  • the mRNA was dissolved in 25 mM sodium acetate buffer solution with a pH of about 5.2 to prepare a nucleic acid preparation with a final concentration of about 0.1 mg/mL.
  • the above prepared liposome raw material solution and nucleic acid preparation are prepared at a two-phase volume ratio of about 4:1, and the total rate of the two-phase solution is 12mL/min, through the Nano Assemblr microfluidic system, or by vortexing Quickly mix the two-phase solution to form uniform and stable mRNA nano-liposome particles, and then rapidly change the environment of the mRNA nano-liposome particles from pH 5.2 to 7.0-7.4.
  • the specific operation is to dilute it to 20 times the volume with PBS buffer solution with pH 7.2 or sodium acetate buffer solution with pH 7.4, and then use a 10kD ultrafiltration tube to concentrate.
  • the speed of the centrifuge should not exceed the maximum speed of the ultrafiltration tube.
  • the pH of the solution environment of the mRNA nanoliposome particles is about 7.2 to 7.4, concentrate it to a final concentration of about 200mM, and place it at 4°C for standby or add a stabilizer to freeze. Stored at -70 degrees.
  • the particle size, PDI, encapsulation efficiency and potential of the mRNA nanolipid particles were tested by the same detection method as that of the pDNA nanolipid particles, and the test results are shown in Table 2 and FIG. 13 .
  • Table 2 and Figure 13 show that the average particle size of mRNA nano liposome particles Lipid-1, Lipid-2 and Lipid-3 is all at 100-150nm, the particle size distribution is uniform, the encapsulation efficiency is all above 90%, and the potential is -20.8 ⁇ -10.2mV.
  • Example 5 Gel retardation experiments were performed to detect the encapsulation effect of mRNA nanoliposome particles on mRNA and the encapsulation effect of pDNA nanoliposome particles on pDNA under different N:P ratios.
  • mRNA is unstable and is particularly susceptible to degradation by RNA hydrolase. Therefore, as a transfection reagent for mRNA, it is currently necessary to encapsulate mRNA to play a protective role.
  • MC3 liposome adopts Dlin-MC3-DMA
  • MC3 liposome adopts Dlin-MC3-DMA
  • Example 6 In vitro transfection experiments of suspension cells 293T cells, Hela cells and Jurkat cells using mRNA nanoliposome particle Lipid-1.
  • the average fluorescence intensity of mRNA lipid nanoparticle Lipid-1 transfection 293T cells and Hela cells is shown in Figure 16 (in Fig. The average fluorescence intensity in the cell), the in vitro transfection fluorescence micrograph of the suspension cell line Jurkat cells by mRNA lipid nanoparticle Lipid-1 and the proportion of positive cells are shown in Figure 17, the results show that the mRNA lipid nanoparticle Lipid-1 is suitable for suspension Transfection of cell lines.
  • Example 7 In vitro transfection experiments of various adherent cells using pDNA nanoliposome particle Lipid-1.
  • the culture was continued for 24 hours, and the transfection effect of the liposomes on each cell was observed with an inverted fluorescence microscope, as shown in Figure 18, the results indicated that the pDNA nanoliposome particle Lipid-1 was suitable for the transfection of various types of cells.
  • Example 8 Stability testing experiment of mRNA nano-lipid particles in the environment of RNase.
  • Encapsulated mRNA (referred to as Free mRNA) was used as a control.
  • the nano-lipid particles were evenly added to the centrifuge tubes, and the amount of mRNA contained in each centrifuge tube was 2ug.
  • the Free-mRNA was added to another new series of centrifuge tubes according to the dose of 2ug. Add RNase A in different concentrations to each centrifuge tube, disperse evenly, and place at room temperature for 1 hour.
  • Example 9 In vitro transfection cytotoxicity test experiment.
  • the synthesized lipid molecules are designed with water-transporting tails that can be degraded by cellular metabolism of ester groups.
  • the lactate dehydrogenase cytotoxicity detection kit is used, and the specific steps are as follows: inoculate cells with an appropriate density into a 96-well plate so that the density of the cells to be tested does not exceed 80%, and add different amounts of Lipid-1. After the incubation, centrifuge, remove the supernatant, add LDH release reagent diluted 10 times with 150 ⁇ L PBS, mix well, incubate for 1 hour, centrifuge, take the supernatant, and measure the absorbance. The cytotoxicity results of liposomes are shown in Figure 20. . With the increase of the liposome concentration, the survival rate of the cells remained above 98%, indicating that the nano-lipid particles had no obvious cytotoxicity.

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Abstract

提供了一种可离子化脂质化合物及核酸体外细胞转染试剂。

Description

可离子化脂质化合物及核酸体外细胞转染试剂
相关申请的交叉引用
本申请要求在2021年12月15日在中国提交的中国专利申请号202111534575.8的优先权，其全部内容通过引用并入本文。
技术领域
本公开属于体外细胞转染试剂技术领域，具体涉及可离子化脂质化合物及核酸转染试剂。
背景技术
细胞转染是指将外源分子如DNA，RNA等导入真核细胞的技术。随着分子生物学和细胞生物学研究的不断发展，转染已经成为研究和控制真核细胞基因功能的常规工具。在研究基因功能、调控基因表达、突变分析和蛋白质生产等生物学试验中，其应用越来越广泛。
以往，由于mRNA难以成药的特点，在体外将mRNA转入细胞的应用相对于DNA的转染应用非常少。如今，mRNA药物迅速发展，体外转染mRNA进入细胞的需求快速增长，但是可用的转染试剂屈指可数。科研与生产急需开发适用于mRNA的体外转染试剂。
目前，常用的DNA细胞转染方法主要分为三类途径：物理介导(电穿孔法，基因枪法、显微注射法)、化学介导(脂质体转染法、磷酸钙共沉淀法、阳离子聚合物介导法)、生物介导(病毒介导转染、原生质体转染)。电穿孔法的缺点是细胞致死率高；显微注射和基因枪的缺点是操作复杂、所需设备昂贵且会导致核酸片段缺失或突变；原生质体转染的缺点是转染效率低且成本高；磷酸钙共沉淀法的缺点是对DNA浓度要求高且适用性有局限；阳离子脂质体转染法对DNA浓度有一定要求且对细胞有一定的毒性。病毒介导的转染技术，虽然转染效率较高，同时具有低细胞毒性的优势，但是，病毒转染方法的准备程序复杂，常常对细胞类型有很强的选择性，在一般实验室中很难普及。
现有的商业化体外细胞脂质转染试剂多数为阳离子脂质体，为带正电荷的脂质体，其靠静电作用和DNA结合形成DNA-脂质体复合物，然后通过细胞的内吞作用进入细胞，具有操作简单、适用性广等优势，但是对不同细胞的转染效率还有待进一步提高、对细胞仍然存在一定的毒性。理想细胞转染试剂，应该具有转染效率高、细胞毒性小等优点。虽然现有阳离子脂质体转染试剂在包封效率、转染效率和细胞毒性等方面相比病毒载体和其他类型的非病毒载体都具有明显的优势，但是依然存在一定的细胞毒性，转染效率也需进一 步提高，因此，需要开发更多转染效率高且细胞毒性低的体外细胞转染试剂，为细胞转染提供更多的选择。
发明内容
本公开的目的是提供一种易与DNA以及RNA结合且易降解的可离子化脂质化合物，其丰富了可离子化脂质化合物的种类，可用于制备DNA体外细胞转染试剂或RNA体外细胞转染试剂，为细胞体外DNA转染或RNA转染提供更多的选择。
本公开的另一目的是提供一种转染效率高且细胞毒性小的细胞体外核酸转染试剂，可用于体外细胞体外DNA转染或RNA转染。
一种可离子化脂质化合物，所述可离子化脂质化合物为通式(Ⅰ)、通式(Ⅱ)、通式(Ⅲ)所示的化合物中的一种或多种：
Figure PCTCN2022121381-appb-000001
其中，
R为-OC(＝O)-或-C(＝O)O-。
本公开的实施例中，R的结构以其所在链的靠近N的一端向另一端的方向进行限定的，即当R为-OC(＝O)-，靠近N的是酯基中的O，当R为-C(＝O)O-时，靠近N的是酯基中的C。
R 1为氢、甲基、乙基或异丙基。
m为1～10之间的整数，例如m为1、2、3、4、5、6、7、8、9或10。
n为1～3之间的整数，例如n为1、2或3。
f为1～5之间的整数，例如f为1、2、3、4或5。
x为1～8之间的整数，例如x为1、2、3、4、5、6、7或8。
y为1～9之间的整数，例如y为1、2、3、4、5、6、7、8或9。
R 2和R 3独立地为氢、甲基、乙基或异丙基。
p为1～5之间的整数，例如p为1、2、3、4或5。
q为1～3之间的整数，例如q为1、2或3。
在一些实施例中，所述的R为-C(＝O)O-。
在一些实施例中，所述的R 1为氢。
在一些实施例中，所述的R 2和所述的R 3中一个为氢，另一个为甲基、乙基或异丙基。
在一些实施例中，所述的R 2和所述的R 3中一个为氢，另一个为甲基。
在一些实施例中，所述的m为3～8之间的整数。在另一些实施例中，m为4～6之间的整数。
在一些实施例中，所述的f为1～4之间的整数。在另一些实施例中，f为2或3。
在一些实施例中，所述的x为2～5之间的整数。在另一些实施例中，x为2～4之间的整数。
在一些实施例中，所述的y为3～9之间的整数。在另一些实施例中，y为5～9之间的整数。
在一些实施例中，所述的R为-C(＝O)O-，所述的R 1为氢，所述的n为2，所述的R 2和所述的R 3中一个为氢，另一个为甲基，所述的p为5。
根据一些实施例，通式(I)中，R 1为氢，m为4～6之间的整数，n为2，f为1～3之间的整数。
根据一些实施例，通式(Ⅱ)中，x为3，y为6～9之间的整数。
根据一些实施例，通式(Ⅲ)中，R 2为甲基，R 3为氢，p为5，q为1～3之间的整数。
根据一些实施方式，所述的可离子化脂质化合物为如下结构式所示化合物中的一种或多种：
Figure PCTCN2022121381-appb-000002
一种核酸体外细胞转染试剂，所述的核酸体外细胞转染试剂包括权利要求1至7中任一项所述的可离子化脂质化合物与辅助脂质分子形成的组合物。
在一些实施例中，所述的核酸包括脱氧核糖核酸和核糖核酸，所述的脱氧核糖核酸为质粒DNA，核糖核酸为mRNA。
在一些实施例中，所述的辅助脂质分子包括胆固醇、卡泊三醇、豆甾醇、β-谷甾醇、白桦脂醇、熊果酸、羽扇豆醇、齐墩果酸、二油酰基磷脂酰胆碱、二硬脂酰磷脂酰胆碱、1-硬脂酰基-2-油酰基卵磷脂、二油酰磷脂酰乙醇胺、(1,2-二油氧基丙基)三甲基氯化铵、双十烷基二甲基溴化铵、1,2-二肉豆蔻酰基-sn-甘油-3-乙基磷酸胆碱、二棕榈酰磷脂酰乙醇胺-甲氧基聚乙二醇5000、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、活性炭、二氧化硅以及磷酸钙中的一种或多种中的一种或多种。
在一些实施例中，所述的可离子化脂质化合物与所述的辅助脂质分子的投料摩尔比为1:(0.5～2)。
在一些实施例中，所述的核酸与所述的组合物的质量比为1:(5～50)。进一步优选在一些实施例中为1:(5～40)。在一些实施例中为1:(5～30)。在一些实施例中为1:(5～20)。
在一些实施例中，所述的核酸与所述的组合物的N：P比值为5～10:1。
在一些实施例中，所述的核酸与所述的组合物的N：P比值为5～8:1。
可离子化的脂质分子其组合物最近被发展成为重要的药物递送载体，但是应用于DNA体外细胞的转染的实例还很少，应用于RNA体外细胞的转染的实例还未见报道。我们发现可离子化的脂质分子其组合物作为DNA体外细胞的转染试剂具有明显的毒性小的优势，同时我们发明的一系列可离子化脂质化合物其组合物对于DNA难转染的悬浮细胞例如Jurkat 细胞的转染效率明显高于常用的其它脂质转染试剂lipo2000。可离子化脂质化合物及其组合物在DNA体外细胞转染以及mRNA体外细胞转染领域的应用将促进真核细胞基因功能研究和广泛应用。
本公开的实施例与现有技术相比具有如下优势：
本公开的实施例提供了一种新的可离子化脂质化合物，丰富了可离子化脂质化合物种类，为脱氧核糖核酸以及核糖核酸转染体外细胞提供更多选择，其形成的脂质体颗粒大小均匀、稳定性好、包封效率高、递送效率高且毒性低，可作为体外细胞DNA转染及RNA转染的转染试剂，对相关领域的发展和应用具有重要的意义。
附图说明
图1为化合物1-1的氢谱图；
图2为2-(双(2-氨基乙基)氨基)乙-1-醇)的氢谱图；
图3为化合物1的氢谱图；
图4为化合物1的质谱图；
图5为化合物2-1的氢谱图；
图6为化合物2的氢谱图；
图7为化合物2的质谱图；
图8为化合物3-1的氢谱图；
图9为1,3-二氨基-2-丙醇的氢谱图；
图10为化合物3的氢谱图；
图11为化合物3的质谱图；
图12为pDNA脂质纳米颗粒Lipid-1的透射电镜图以及zeta电位测试结果图；
图13为mRNA脂质纳米颗粒的粒径分布图；
图14为mRNA脂质纳米颗粒Lipid-3的凝胶阻滞实验电泳图；
图15为不同pDNA与脂质分子用量比例下的pDNA脂质纳米颗粒的凝胶阻滞实验电泳图；
图16为mRNA脂质纳米颗粒Lipid-1转染293T细胞和Hela细胞的平均荧光强度随时间的变化；
图17为mRNA脂质纳米颗粒Lipid-1对悬浮细胞系Jurkat细胞的体外转染荧光显微镜图及阳性细胞占比；
图18为pDNA脂质纳米颗粒Lipid-1对多种贴壁细胞(MCF-7细胞、Hela细胞及293T细胞)进行体外转染的荧光显微镜图；
图19为mRNA纳米脂质颗粒在RNA水解酶的环境下的稳定性测试实验结果图；
图20为脂质纳米颗粒Lipid-1体外转染293T细胞的毒性测定试验结果图。
具体实施方式
下面结合实施例对本公开作进一步描述。但本公开并不限于以下实施例。实施例中采用的实施条件可以根据具体使用的不同要求做进一步调整，未注明的实施条件为本行业中的常规条件。本公开各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
当前，基于脂质体转染载体结构简单、操作方便、生物安全性高等优点，其已成为目前应用最为广泛的非病毒载体，但是大多脂质体载体转染效率仍然不够高，核酸体内表达效果不好，而且对细胞具有一定的毒性，从而限制了脂质体载体的更广泛的应用。因此，为了提高脂质体的递送效率，同时降低细胞毒性，发明人经过大量的研究和实验，开发了一种新的可离子化脂质化合物及包括该可离子化脂质化合物的体外细胞转染试剂。
通常将可离子化脂质分子分为三个部分，包括头基、连接基和尾基。头基(Head)在弱酸性的环境下能够发生质子化，使可离子化脂质整体带有正电荷，使可离子脂质分子与显负电性的核酸结合成紧实的球状结构，即形成纳米脂质颗粒，纳米脂质颗粒在进入细胞后先进入早期内体，然后进入晚期内体，最后进入溶酶体，其中大部分的核酸会被内质网中的酶降解。晚期内涵体酸性降低，会促进脂质纳米粒质子化，破坏内体膜，部分脂质纳米颗粒能够通过内体逃逸而使核酸免受降解，因此避免了核酸过早降解。连接基(Linker)是另一个重要的功能部分，用以连接可离子化脂质分子的头基和尾基。该部位不仅会影响脂质体的整体pKa，还会影响脂质体的电位及生物降解性。尾基(Tail)是第三重要的功能部分，通常具有长的疏水碳链而使脂质长链具有“脂质性质”。因此，头基、连接基和尾基的结构都对脂质体的转染效率、毒性程度等有关。
发明人从头基、连接基和尾基三个部分进行了充分的研究，最终提供了通式(Ⅰ)、通式(Ⅱ)或通式(Ⅲ)所示的可离子化脂质化合物，
Figure PCTCN2022121381-appb-000003
Figure PCTCN2022121381-appb-000004
其中，
R为-OC(＝O)-或-C(＝O)O-；
R 1为氢、甲基、乙基或异丙基；
m为1～10之间的整数；
n为1～3之间的整数；
f为1～5之间的整数；
x为1～8之间的整数；
y为1～9之间的整数；
R 2和R 3独立地为氢、甲基、乙基或异丙基；
p为1～5之间的整数；
q为1～3之间的整数。
本公开实施例的可离子化脂质化合物中的多个叔胺基团及特殊的对称性头基结构，在酸性环境下质子化而带正电荷，提高了与带负电荷的核酸的结合能力，并防止核酸在细胞内过早被核酸水解酶降解。
在一些实施例中，R为-C(＝O)O-。
对于通式(Ⅰ)，在一些实施例中，R 1为氢，m为1～10之间的整数，n为1～3之间的整数，f为1～5之间的整数；在另一些实施例中，R 1为氢，m为3～8之间的整数，n为1～3之间的整数，f为1～4之间的整数；在另一些实施例中，R 1为氢，m为4～6之间的整数，n为1～3之间的整数，f为1～3之间的整数；在另一些实施例中，R 1为氢，m为4～6之间的 整数，n为2，f为1～3之间的整数。
对于通式(Ⅱ)，在一些实施例中，x为1～6之间的整数，y为3～9之间的整数；在另一些实施例中，x为2～5之间的整数，y为5～9之间的整数；在另一些实施例中，x为2～4之间的整数，y为6～9之间的整数；在另一些实施例中，x为3，y为6～9之间的整数。
对于通式(Ⅲ)，在一些实施例中，R 2和R 3独立地为氢或甲基，p为1～5之间的整数，q为1～3之间的整数；在另一些实施例中，R 2为甲基，R 3为氢，p为1～5之间的整数，q为1～3之间的整数；在另一些实施例中，R 2为甲基，R 3为氢，p为2～5之间的整数，q为1～3之间的整数；在另一些实施例中，R 2为甲基，R 3为氢，p为5，q为1～3之间的整数。
合适的尾基结构能够提高核酸传递系统的效率，本公开的实施例的可离子化脂质化合物具有多条相同的烃基尾部，有利于装载核酸的脂质体穿越细胞膜，适用于多种类型细胞的转染，核酸递送效率与现有已商品化转染试剂相当或更高，结合其连接基的特殊结构设计，能够实现有效降解，从而实现细胞内快速清除，降低了脂质体的毒副作用。
本公开实施例的可离子化脂质化合物具有以下优点：
(1)本公开实施例的可离子化脂质化合物形成的脂质体，在pH＝7～8的缓冲环境下成电中性，在酸性的缓冲环境中带正电，能够有效地与显负电荷的DNA和RNA紧密结合成纳米颗粒，颗粒大小均匀，稳定性好且包封效率高。
(2)本公开实施例的可离子化脂质化合物形成的脂质体，在晚期内涵体酸性环境下带正电荷，从而可以与内涵体膜中的磷脂阴离子作用，形成适应非双层结构的离子对，继而破坏内涵体膜，实现内涵体逃逸突破细胞内的障碍。因此，可有效地将核酸递送到细胞质中，后续实现有效基因表达。
(3)本公开实施例的可离子化脂质化合物的连接基为经设计的带酯基基团，使脂质体容易降解，降低了细胞毒性。
(4)本公开实施例的可离子化脂质化合物形成的脂质体，在多种类型的细胞中都表现出较好的转染效率，表明其作为核酸普通转染试剂应用的有效性和可转染悬浮细胞独特性。
(6)本公开实施例中可离子化脂质化合物的合成步骤简单，反应条件温和、成本较低，值得推广利用。
下面根据一些具体实施例，进一步说明本公开实施例的技术方案和有益效果。
下述实施例中的实验方法，如无特殊说明，均为常规方法；所用的实验材料，如无特殊说明，均为常规生化试剂厂商购买得到的。
以下实施例中，Lipid-1仅是指使用实施例1制备的化合物1作为可电离脂质化合物与其他组分形成的纳米脂质颗粒，Lipid-2仅是指使用实施例2制备的化合物2作为可电离脂质化合物与其他组分形成的纳米脂质颗粒，Lipid-3仅是指使用实施例3制备的化合物3作 为可电离脂质化合物与其他组分形成的纳米脂质颗粒，对各纳米脂质颗粒中包封的核酸类型没有限制。
实施例1
化合物1的合成路线：
Figure PCTCN2022121381-appb-000005
步骤1：化合物1-1的合成：
将亚麻醇(0.267g，1mmol)和三乙胺(0.133g，1.3mmol)加入反应瓶中冰水浴，加入二氯甲烷(6mL)，将丙烯酰氯(0.11g，1.2mmol)溶于二氯甲烷(2.2mL)，缓慢滴加入反应瓶中，反应持续10分钟，该反应保持在10℃以下，最后冰浴移除，反应液在室温条件下反应2小时。用饱和食盐水洗涤，得到粗产物，将粗产品通过色谱法(硅胶柱，洗脱剂为含有0.5％EA(体积百分比)的石油醚)纯化，并将纯产物蒸发，得到浅黄色油状化合物1-1(2-烯丙酸(9Z，12Z)-十八碳二烯酯)(0.173g，收率：50％)化合物1-1的氢谱见图1。
1H NMR(400MHz,CDCl3)δ:6.41(dd,J＝17.3,1.5Hz,1H),6.13(dd,J＝17.3,10.4Hz,1H),5.82(dd,J＝10.4,1.5Hz,1H),5.47-5.26(m,4H),4.16(t,J＝6.7Hz,2H),2.78(t,J＝6.5Hz,2H),2.06(dd,J＝13.6,6.7Hz,4H),1.75-1.60(m,2H),1.39-1.17(m,16H),0.88(dt,J＝10.4,5.3Hz,3H).
步骤2：化合物1的合成：
将2-(双(2-氨基乙基)氨基)乙-1-醇)(0.0735g，0.50mmol，氢谱见图2)和2-烯丙酸(9Z，12Z)-十八碳二烯酯(0.64g，2mmol)加入反应瓶中，在80℃下反应48小时。反应冷却至室温后，真空除去溶剂，得到粗产品，将粗产品通过色谱法(硅胶柱，洗脱剂为含有0.5％甲醇(体积百分比)二氯甲烷纯化，并将纯产物蒸发，得到黄色油状化合物1(25.7mg，收率：3.6％)。化合物的氢谱见图3，质谱见图4。
1H NMR(400MHz,CDCl3)δ5.45-5.30(m,16H),4.07(t,J＝6.8Hz,8H),3.52(s,1H),2.80(dd,J＝12.5,6.4Hz,16H),2.63(s,4H),2.54(s,3H),2.48(t,J＝7.2Hz,8H),2.07(q,J＝6.7Hz,16H),1.62(dd,J＝13.4,6.6Hz,8H),1.42-1.25(m,68H),0.91(t,J＝6.8Hz,12H),0.91(t,J＝6.8Hz,1H)。
实施例2
化合物2的合成路线
步骤1：化合物2-1的合成
Figure PCTCN2022121381-appb-000006
将6-溴己酸(1.0g，5.13mmol)和十一醇(1.77g，10.25mmol)溶于二氯甲烷中(60mL)，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC盐酸盐，0.98g，5.13mmol)和DMAP(0.125g，1.03mmol)。混合物在常温条件下搅拌18小时。反应结束后，用DCM(200mL)稀释，并用饱和的NaHCO3(100mL)和盐水(100mL)洗涤。合并有机层用无水Na2SO4干燥，真空除去溶剂，得到粗产品，将粗产品通过色谱法(硅胶柱，洗脱剂为含有0.5％EA(体积百分比)的石油醚)纯化，并将纯产物蒸发，得到浅黄色油状化合物2-1(6-溴己酸十一酯)(0.69g，收率38.6％)。化合物2-1的氢谱见图5。
1H NMR(400MHz,CDCl3)δ:4.10(t,J＝6.6Hz,2H),3.45(t,J＝6.7Hz,2H),2.36(t,J＝7.3Hz,2H),1.97-1.88(m,2H),1.68(tt,J＝14.5,7.3Hz,4H),1.53(dd,J＝15.1,7.9Hz,2H),1.33(d,J＝16.9Hz,16H),0.92(t,J＝6.5Hz,3H).
步骤2：化合物2的合成
将2-(双(2-氨基乙基)氨基)乙-1-醇)(0.044g，0.30mmol)和6-溴己酸十一酯(0.417g，1.20mmol)溶于THF/CH3CN(1:1，6mL)，之后再加入DIPEA(0.155g，1.20mmol)。反应物在63℃条件下搅拌72h，反应冷却至室温后，真空除去溶剂。粗产品用乙酸乙酯和饱和的NaHCO3萃取，合并有机层用无水Na2SO4干燥，真空除去溶剂，得到粗产品，将粗产品通过色谱法(硅胶柱，洗脱剂为含有1-2％甲醇(体积百分比)二氯甲烷)纯化，并将纯产物蒸发，得到黄色油状化合物2(14.64mg，收率4％)。化合物2的氢谱见图6，质谱见图7。
1H NMR(400MHz,CDCl3)δ4.10-3.98(m,8H),3.63(d,J＝16.5Hz,2H),3.46(s,1H),3.22(d,J＝45.3Hz,3H),3.05(d,J＝4.0Hz,2H),3.01-2.73(m,9H),2.66(d,J＝14.3Hz,2H),2.33(dd,J＝16.6,7.4Hz,8H),1.98(s,2H),1.78(s,2H),1.64(dt,J＝23.5,7.0Hz,20H),1.41-1.20(m,74H),0.88(t,J＝6.7Hz,12H)。
实施例3
化合物3的合成路线
Figure PCTCN2022121381-appb-000007
步骤1：化合物3-1的合成
将8-溴辛酸(1.139g，5.13mmol)和3,7-二甲基辛-6-烯-1-醇(香茅醇，1.599g，10.25mmol)溶于二氯甲烷(60mL)，充分溶解后，加入EDC盐酸盐(0.98g，5.13mmol)和DMAP(0.125g，1.03mmol)。混合物在常温条件下搅拌18小时。反应结束后，用DCM(200mL)稀释，并用饱和的NaHCO3(100mL)和盐水(100mL)洗涤。合并有机层用无水Na2SO4干燥，真空除去溶剂，得到粗产品,将粗产品通过色谱法(硅胶柱，洗脱剂为含有0.5％EA(体积百分比)的石油醚)纯化，并将纯产物蒸发，得到浅黄色油状化合物3-1(6-溴己酸3,7-二甲基辛-6-烯酯)(0.648g，35％)化合物3-1的氢谱见图8。
1H NMR(400MHz,CDCl3)δ:5.09(s,1H),4.18-4.01(m,2H),3.40(t,J＝6.8Hz,2H),2.29(t,J＝7.4Hz,2H),1.98(s,2H),1.84(dd,J＝14.3,7.0Hz,2H),1.70-1.60(m,9H),1.38(d,J＝37.7Hz,9H),0.89(t,J＝12.9Hz,4H).
步骤2：化合物3的合成
将1,3-二氨基-2-丙醇(0.027g，0.30mmol，氢谱见图9)和6-溴己酸3,7-二甲基辛-6-烯酯(0.398g，1.2mmol)加入反应瓶中溶于THF/CH3CN(1:1，6mL)，之后再加入DIPEA(0.155g，1.20mmol)。反应物在63℃条件下搅拌72h，反应冷却至室温后，真空除去溶剂。粗产品用乙酸乙酯和饱和的NaHCO3萃取，合并有机层用无水Na2SO4干燥，真空除去溶剂，得到粗产品，将粗产品通过色谱法将粗产品通过色谱法(硅胶柱，洗脱剂为含有1％甲醇(体积百分比)二氯甲烷)纯化，并将纯产物蒸发，得到浅黄色油状化合物3(11.63mg，收率3.2％)。化合物3的氢谱见图10，质谱见图11。
1H NMR(400MHz,CDCl3)δ5.30(s,1H),5.08(t,J＝6.4Hz,4H),4.10(h,J＝10.9Hz,8H),3.67(s,1H),2.46(s,10H),2.28(t,J＝7.3Hz,8H),1.97(d,J＝9.2Hz,8H),1.74-1.51(m, 41H),1.49-1.37(m,12H),1.37-1.16(m,33H),0.91(d,J＝5.9Hz,12H)。
实施例4：纳米脂质颗粒的制备、粒径及电位的测定：
使用无水乙醇作为溶剂，将实施例1至3中制备的化合物1、化合物2以及化合物3分别与DSPC、DMG-PEG2000及胆固醇按照摩尔比为50:10:1.5:38.5溶解混合得到脂质体原料溶液，控制各组分浓度之和为50mM，完全溶解混匀后放置-20℃保存。
将pDNA分散于25mM的醋酸钠缓冲液中，加入上述脂质体原料溶液共孵育20分钟，使脂质体与pDNA紧密结合形成pDNA纳米脂质颗粒，分别标记为Lipid-1、Lipid-2和Lipid-3，合格的脂质纳米颗粒就可以用来转染准备好的细胞。利用Zetasizer Nano ZS(Malvern,Worcestershire,UK)检测纳米脂质体颗粒的粒径，PDI。粒径的测量通过将纳米脂质体颗粒溶液用1×PBS稀释50倍之后测量，Zeta电位的测定是通过将纳米脂质体颗粒稀释到15mM的PBS中进行测定，检测结果如表1和图12所示。
表1 脂质分子包封pDNA形成的纳米粒的各项参数
Figure PCTCN2022121381-appb-000008
表1和图12显示，pDNA纳米脂质颗粒Lipid-1、Lipid-2和Lipid-3的平均粒径在120-150nm范围内，粒径分布均匀，包封效率均在90％以上，电位在-18.6～-11.6mV。
将mRNA溶解在pH为5.2左右的25mM的醋酸钠缓冲液中，制得终浓度约为0.1mg/mL的核酸制剂。将上述配制的脂质体原料溶液和核酸制剂以两相体积比约为4:1，两相溶液总速率为12mL/min条件下，通过Nano Assemblr微流控系统，也可通过涡旋的方法快速地将两相溶液混匀，形成均一稳定的mRNA纳米脂质体颗粒，然后快速地将mRNA纳米脂质体颗粒环境从pH为5.2转变成7.0～7.4。具体操作为，用pH为7.2的PBS缓冲液或pH为7.4的醋酸钠缓冲液将其稀释20倍体积后，利用10kD的超滤管进行浓缩，离心机的转速不要超过超滤管的最高转速限制，经过2～3次换液之后，mRNA纳米脂质体颗粒的溶液环境的pH在7.2～7.4左右，将其浓缩至终浓度为200mM左右，放置在4℃环境下备用或添 加稳定剂冻存于-70度。
采用与pDNA纳米脂质颗粒相同的检测方法测试mRNA纳米脂质体颗粒的粒径、PDI、包封率及电位，测试结果见表2和图13。
表2 脂质分子包封mRNA形成的纳米粒的各项参数
Figure PCTCN2022121381-appb-000009
表2及图13显示，mRNA纳米脂质体颗粒Lipid-1、Lipid-2和Lipid-3的平均粒径都在100-150nm，粒径分布均匀，包封效率均在90％以上，电位为-20.8～-10.2mV。
实施例5.凝胶阻滞实验检测mRNA纳米脂质体颗粒对mRNA的包封效果以及不同N：P比下pDNA纳米脂质体颗粒对pDNA的包封效果。
与pDNA不同，mRNA不稳定，特别容易受到RNA水解酶的降解，所以作为mRNA的转染试剂目前必须要将mRNA包封起来，以起到保护作用。我们通过凝胶阻滞实验重点比较Lipid-1、Lipid-2以及Lipid-3与参照物MC3(脂质体采用Dlin-MC3-DMA)对mRNA的包封效果，其琼脂糖凝胶电泳结果见图14。每一胶条右边的泳道里核酸分子被脂质体阻截在上样孔里，不能在琼脂糖凝胶里向下迁移，而左边泳道里的marker可以正常迁移。说明所有mRNA纳米脂质体颗粒对mRNA的包封效果很好。
为了探究脂质体与pDNA的结合能力，我们将不同N：P比的脂质体和pDNA分别在pH＝5.2的醋酸钠缓冲液中孵育20min之后，通过DNA琼脂糖凝胶观察脂质体和pDNA的结合情况，结果见图15，最左侧泳道为DNA mark，第二泳道为pDNA，第三泳道至第第七泳道依次为N：P为3:1、4:1、5:1、6:1、7:1，由图12可见，Lipid-1、Lipid-2和Lipid-3对pDNA的包封效果好，最适N：P比值为7:1
实施例6：采用mRNA纳米脂质体颗粒Lipid-1进行悬浮细胞293T细胞、Hela细胞以及Jurkat细胞的体外转染实验。
选用293T细胞系、Hela细胞系以及Jurkat细胞系，在培养瓶中培养至适量密度，接种到6孔板中，培养24小时之后，将通过体外转录得到的纯净的带有绿色荧光GPF的mRNA和实施例4中的DSPC、DMG-PEG2000及胆固醇在pH＝5.2的醋酸钠缓冲液中孵育20min，使脂质分子与mRNA的N:P为7:1使mRNA充分包封脂质体中，随后将该混合溶液加入到各六孔板中，进行细胞转染。2h后即可观察到绿色荧光，可持续表达长达72h。mRNA脂质纳米颗粒Lipid-1转染293T细胞和Hela细胞的平均荧光强度随时间的变化见图16(图16中每组左侧柱形表示293T细胞中平均荧光强度，右侧柱形表示Hela细胞中平均荧光强度)，mRNA脂质纳米颗粒Lipid-1对悬浮细胞系Jurkat细胞的体外转染荧光显微镜图及阳性细胞占比见图17，结果表明mRNA脂质纳米颗粒Lipid-1适用于悬浮细胞系的转染。
实施例7：采用pDNA纳米脂质体颗粒Lipid-1进行多种贴壁细胞的体外转染实验。
在转染细胞之前，将培养瓶中适量密度的Hela细胞、MCF-7细胞以及293T细胞转移到6孔板中，培养12小时之后，将带有绿色荧光GPF标签的质粒和实施例4中的DSPC、DMG-PEG2000及胆固醇在pH＝5.2的醋酸钠缓冲液中孵育20min，使质粒与脂质体以N:P比为7:1的比例充分结合，随后将该混合溶液加入到各六孔板中，进行细胞转染。4-6h后，去上清，换成新鲜的带血清的DMEM培养基，目的是将转染试剂去除。继续培养24小时，用倒置荧光显微镜观察脂质体对各细胞的转染效果，如图18所示，该结果表明pDNA纳米脂质体颗粒Lipid-1适用于多种类型细胞的转染。
实施例8：mRNA纳米脂质颗粒在RNA酶的环境下的稳定性测试实验。
为了探究我们的脂质体包封mRNA之后形成的纳米脂质颗粒是否能够保护mRNA免受RNA酶的降解，在实验之前，我们将mRNA包封成纳米脂质颗粒，并将未被脂质体包封的mRNA(简称Free mRNA)作为对照。首先将纳米脂质颗粒平均加入到离心管中，每只离心管中含有的mRNA的量为2ug，同时也将Free-mRNA按照2ug的剂量加入另外新的一系列离心管中。将RNase A以不同的浓度加入到各个离心管中，分散均匀，室温下放置1小时。最后通过琼脂糖凝胶电泳及胶图成像探究mRNA的降解情况。实验结果如图19所示，该结果表明，Free mRNA在RNase A的环境下能够被降解，而脂质体包封后的mRNA在RNase A浓度较高的的环境下能够稳定免受降解。
实施例9：体外转染细胞毒性测试实验。
所合成的脂质分子设计了其输水尾部可以通过酯基的细胞代谢来降解。采用乳酸脱氢酶细胞毒性检测试剂盒，具体步骤：将适量密度的细胞接种到96孔板中，使待测细胞的密 度不超过80％，加入不同量的Lipid-1。孵育结束后，离心，去除上清液，加入150μL PBS稀释了10倍的LDH释放试剂，混匀，孵育1小时，离心，取上清，测定吸光度，脂质体的细胞毒性结果如图20示。随着脂质体浓度的增加，细胞的存活率均保持在98％以上，表明纳米脂质颗粒没有明显的细胞毒性。
以上对本公开实施例做了详尽的描述，其目的在于让熟悉此领域技术的人士能够了解本公开实施例的内容并加以实施，并不能以此限制本公开的保护范围，凡根据本公开的精神实质所作的等效变化或修饰，都应涵盖在本公开的保护范围内。

Claims (11)

  1. 一种可离子化脂质化合物，其特征在于：所述可离子化脂质化合物为通式(Ⅰ)、通式(Ⅱ)、通式(Ⅲ)所示的化合物中的一种：
    Figure PCTCN2022121381-appb-100001
    其中，
    R为-C(＝O)O-，R的结构以其所在链的靠近N的一端向另一端的方向进行限定的，靠近N的是酯基中的C；
    R 1为氢、甲基、乙基或异丙基；
    m为1～10之间的整数；
    n为1～3之间的整数；
    f为1～5之间的整数；
    x为1～8之间的整数；
    y为1～9之间的整数；
    R 2和R 3独立地为氢、甲基、乙基或异丙基；
    p为1～5之间的整数；
    q为1～3之间的整数。
  2. 根据权利要求1所述的可离子化脂质化合物，其特征在于，所述的R 1为氢。
  3. 根据权利要求1所述的可离子化脂质化合物，其特征在于，所述的R 2和所述的R 3中一个为氢，另一个为甲基、乙基或异丙基。
  4. 根据权利要求1所述的可离子化脂质化合物，其特征在于，所述的m为3～8之间的整数；和/或，所述的f为1～4之间的整数；和/或，所述的x为2～5之间的整数；和/或，y为3～9之间的整数。
  5. 根据权利要求1所述的可离子化脂质化合物，其特征在于，所述的R 1为氢，所述的n为2，所述的R 2和所述的R 3中一个为氢，另一个为甲基，所述的p为5。
  6. 根据权利要求1所述的可离子化脂质化合物，其特征在于，所述的可离子化脂质化合物为如下结构式所示化合物中的一种：
    Figure PCTCN2022121381-appb-100002
  7. 一种核酸体外细胞转染试剂，其特征在于，所述的核酸体外细胞转染试剂包括权利要求1至6中任一项所述的可离子化脂质化合物与辅助脂质分子形成的组合物。
  8. 根据权利要求7所述的核酸体外细胞转染试剂，其特征在于，所述的核酸包括脱氧核糖核酸和核糖核酸，所述的脱氧核糖核酸为质粒DNA，核糖核酸为mRNA。
  9. 根据权利要求7所述的核酸体外细胞转染试剂，其特征在于，所述的辅助脂质分子包括胆固醇、卡泊三醇、豆甾醇、β-谷甾醇、白桦脂醇、熊果酸、羽扇豆醇、齐墩果酸、二油酰基磷脂酰胆碱、二硬脂酰磷脂酰胆碱、1-硬脂酰基-2-油酰基卵磷脂、二油酰磷脂酰乙醇胺、(1,2-二油氧基丙基)三甲基氯化铵、双十烷基二甲基溴化铵、1,2-二肉豆蔻酰基-sn- 甘油-3-乙基磷酸胆碱、二棕榈酰磷脂酰乙醇胺-甲氧基聚乙二醇5000、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000中的一种或多种。
  10. 根据权利要求7所述的核酸体外细胞转染试剂，其特征在于，所述的可离子化脂质化合物与所述的辅助脂质分子的投料摩尔比为1:(0.5～2)。
  11. 根据权利要求7所述的核酸体外细胞转染试剂，其特征在于，所述的核酸与所述的组合物的质量比为1:(5～50)。
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