技术领域technical field
本发明属于基因载体制备与应用领域，具体涉及一种阳离子脂质类化合物及其制备方法以及在制备基因载体中的应用。The invention belongs to the field of gene carrier preparation and application, and in particular relates to a cationic lipid compound, its preparation method and its application in the preparation of gene carrier.
背景技术Background technique
核酸转染是指将外源的核酸(包括质粒[plasmid DNA]、小干扰RNA[siRNA]、反义核酸[antisense nucleic acids]、微小RNA[microRNA]等)导入细胞内，使其在细胞内发挥生物学功能，从而达到调控基因表达的目的。Nucleic acid transfection refers to the introduction of exogenous nucleic acid (including plasmid [plasmid DNA], small interfering RNA [siRNA], antisense nucleic acid [antisense nucleic acids], microRNA [microRNA], etc.) Play biological functions, so as to achieve the purpose of regulating gene expression.
基因治疗(gene therapy)是指将目的基因(通常是质粒DNA)导入细胞内，使其表达具有活性的蛋白，从而治疗由基因突变或基因缺陷引起的病变。例如，脂蛋白酶酯酶缺乏症是基因突变引起的；携带野生型脂蛋白酶酯酶基因的腺病毒(AVV)类的基因药物Glybera能够将正常基因传送到肌肉细胞内并表达具有生物活性的LPL蛋白，从而降低血液内的脂肪浓度。Gene therapy refers to the introduction of target genes (usually plasmid DNA) into cells to make them express active proteins, thereby treating diseases caused by gene mutations or gene defects. For example, lipoproteinase esterase deficiency is caused by a genetic mutation; Glybera, an adenovirus (AVV)-like gene drug carrying a wild-type lipoproteinase esterase gene, is capable of delivering the normal gene into muscle cells and expressing the biologically active LPL protein , thereby reducing the fat concentration in the blood.
因为核酸带有强负电荷，而细胞表面也带负电荷，所以裸露的核酸很难穿过细胞膜进入细胞内。要想让核酸进入细胞内发挥作用，必须借助合适的载体。开发安全高效的输送载体是基因治疗(DNA输送)在临床应用的前提。基因载体可以分为病毒载体和非病毒载体两大类。病毒(viral vector)载体容易在体内容易引起机体的免疫原性，还存在着引起插入型基因突变和癌变等潜在隐患。所以，病毒在核酸传送领域的进一步应用研究极大地受到了限制，这使得以非病毒作为核酸传送载体的研究成为必然。非病毒(non-viralvector)载体传送法通常采用的材料包括脂质体，多肽，蛋白质，以及阳离子型高分子等。与病毒传递方法相比较，使用非病毒传送核酸可以避免免疫刺激和癌变等副作用，然而目前非病毒核酸载体普遍存在细胞转染率低、细胞毒性大、价格昂贵等问题。因此，开发高效、低毒、廉价的非病毒基因载体具有重要意义。Because nucleic acids are strongly negatively charged, and the cell surface is also negatively charged, it is difficult for naked nucleic acids to pass through the cell membrane and enter the cell. In order for the nucleic acid to enter the cell and function, it is necessary to use a suitable carrier. The development of safe and efficient delivery vectors is the prerequisite for the clinical application of gene therapy (DNA delivery). Gene vectors can be divided into two categories: viral vectors and non-viral vectors. Viral vectors are easy to cause immunogenicity of the body in the body, and there are potential hidden dangers such as causing insertional gene mutation and carcinogenesis. Therefore, the further application research of viruses in the field of nucleic acid delivery is greatly restricted, which makes it inevitable to use non-viruses as nucleic acid delivery vehicles. Materials commonly used in non-viral vector delivery methods include liposomes, polypeptides, proteins, and cationic polymers. Compared with viral delivery methods, the use of non-viral nucleic acid delivery can avoid side effects such as immune stimulation and carcinogenesis. However, current non-viral nucleic acid vectors generally have problems such as low cell transfection rate, high cytotoxicity, and high price. Therefore, it is of great significance to develop high-efficiency, low-toxicity, and cheap non-viral gene vectors.
发明内容Contents of the invention
针对本领域技术的不足之处，本发明的目的是提供一种细胞毒性低、细胞转染效率高、制备方法简单的基因载体。Aiming at the deficiencies of the technology in the art, the purpose of the present invention is to provide a gene carrier with low cytotoxicity, high cell transfection efficiency and simple preparation method.
本发明的另一目的是提供所述基因载体的制备方法与应用。Another object of the present invention is to provide the preparation method and application of the gene carrier.
实现本发明上述目的的具体技术方案为：一种阳离子脂质类化合物，其化学结构如通式(I)所示：The concrete technical scheme that realizes the above object of the present invention is: a kind of cationic lipid compound, its chemical structure is as shown in general formula (I):
式中R1为where R1 is
R3选自以下基团：R3 is selected from the following groups:
R2选自H、赖氨酸、鸟氨酸、精氨酸或组氨酸的残基；如：R2 is selected from residues of H, lysine, ornithine, arginine or histidine; such as:
X、Y为NH、O或S；n为1或2，m为3或4。X and Y are NH, O or S; n is 1 or 2, and m is 3 or 4.
优选地，本发明的阳离子脂质类化合物为：Preferably, the cationic lipid compound of the present invention is:
优选地，本发明的阳离子脂质类化合物为：Preferably, the cationic lipid compound of the present invention is:
本发明提供了上述化合物在制备基因载体中的应用。The invention provides the application of the above compound in the preparation of gene carrier.
具体地，是将本发明的化合物与DOPE或DOPC或卵磷脂按3～1：1～3的摩尔比例混合，溶解在氯仿中，用氮气流吹干后真空干燥过夜。加入pH7.2的PBS，用Avanti Mini-Extruder脂质体挤出器制备得到载体，在4℃冰箱内保存。Specifically, the compound of the present invention is mixed with DOPE or DOPC or lecithin in a molar ratio of 3-1:1-3, dissolved in chloroform, blown dry with nitrogen flow, and then vacuum-dried overnight. Add PBS with pH 7.2, prepare the carrier with an Avanti Mini-Extruder liposome extruder, and store it in a refrigerator at 4°C.
本发明提供了上述化合物在制备基因治疗药物中的应用。The invention provides the application of the above compound in the preparation of gene therapy medicine.
含有上述化合物的基因载体属于本发明的保护范围。The gene carrier containing the above compounds belongs to the protection scope of the present invention.
本发明提供了含有上述化合物的基因载体在核酸转染中的应用。The invention provides the application of the gene carrier containing the above compound in nucleic acid transfection.
本发明提供了上述化合物的制备方法，包括以下步骤：The present invention provides the preparation method of above-mentioned compound, comprises the following steps:
(1)化合物I或II的合成：将饱和或不饱和烃基氨或醇、Boc-谷氨酸-OH或Boc-天冬氨酸-OH溶于有机溶剂中，在氮气保护下加入缩合剂，在0～35℃下搅拌12～24h后，蒸馏除去有机溶剂，加过量酸，在室温搅拌1～2h后蒸馏除去溶剂，用硅胶柱层析法分离纯化得到化合物I即谷氨酸的饱和或不饱和烃基取代酯或酰胺，或得到化合物II即天冬氨酸的饱和或不饱和烃基取代酯或酰胺；(1) Synthesis of compound I or II: dissolve saturated or unsaturated hydrocarbon-based ammonia or alcohol, Boc-glutamic acid-OH or Boc-aspartic acid-OH in an organic solvent, add a condensing agent under nitrogen protection, After stirring at 0-35°C for 12-24 hours, distill off the organic solvent, add excess acid, distill off the solvent after stirring at room temperature for 1-2 hours, and separate and purify by silica gel column chromatography to obtain compound I, which is the saturated or glutamic acid Unsaturated hydrocarbyl substituted ester or amide, or obtain compound II i.e. saturated or unsaturated hydrocarbyl substituted ester or amide of aspartic acid;
(2)化合物III或IV的合成：(2) Synthesis of compound III or IV:
1)将Boc-鸟氨酸、Boc-鸟氨酸(Boc)-Osu或Boc-赖氨酸、Boc-赖氨酸(Boc)-OSu在碱性条件下有机溶剂中在0～35℃下搅拌24～36h，蒸馏除去有机溶剂后用硅胶柱层析法分离纯化得到中间体Boc-鸟氨酸(Boc)-鸟氨酸(Boc)-OH，或Boc-赖氨酸(Boc)-赖氨酸(Boc)-OH；所述的Boc是碳酸二叔丁酯；1) Put Boc-ornithine, Boc-ornithine (Boc)-Osu or Boc-lysine, Boc-lysine (Boc)-OSu in an organic solvent under alkaline conditions at 0 ~ 35 ° C Stir for 24-36 hours, distill off the organic solvent, and then use silica gel column chromatography to separate and purify to obtain the intermediate Boc-ornithine (Boc)-ornithine (Boc)-OH, or Boc-lysine (Boc)-lysine Amino acid (Boc)-OH; The Boc is di-tert-butyl carbonate;
其中m为3或4；where m is 3 or 4;
2)将1)得到的中间体、N-羟基琥珀酰亚胺、缩合剂在有机溶剂中搅拌12h，再加入Boc-鸟氨酸，或Boc-赖氨酸，在0～35℃下继续搅拌12～24h后蒸馏除去有机溶剂后用硅胶柱层析法分离纯化得到化合物III，即Boc-鸟氨酸(Boc)-鸟氨酸(Boc)-鸟氨酸(Boc)-COOH，或得到化合物IV，即Boc-赖氨酸(Boc)-赖氨酸(Boc)-赖氨酸(Boc)-COOH2) Stir the intermediate, N-hydroxysuccinimide, and condensing agent obtained in 1) in an organic solvent for 12 hours, then add Boc-ornithine or Boc-lysine, and continue stirring at 0-35°C After 12 to 24 hours, the organic solvent was distilled off and purified by silica gel column chromatography to obtain compound III, that is, Boc-ornithine (Boc)-ornithine (Boc)-ornithine (Boc)-COOH, or to obtain compound IV, Boc-Lysine (Boc)-Lysine (Boc)-Lysine (Boc)-COOH
其中m为3或4；where m is 3 or 4;
化合物I与化合物III、缩合剂、N,N-二异丙基乙胺溶于干燥的二氯甲烷中，四者的摩尔比为1:1:2:2或Compound I, compound III, condensing agent, and N,N-diisopropylethylamine are dissolved in dry dichloromethane, and the molar ratio of the four is 1:1:2:2 or
化合物II与化合物III、缩合剂、N,N-二异丙基乙胺溶于干燥的二氯甲烷中，四者的摩尔比为1:1:2:2或Compound II, compound III, condensing agent, and N,N-diisopropylethylamine are dissolved in dry dichloromethane, and the molar ratio of the four is 1:1:2:2 or
胆固醇与化合物III、缩合剂、N,N-二异丙基乙胺溶于干燥的二氯甲烷中，四者的摩尔比为2：1：2：2或Cholesterol, compound III, condensing agent, and N,N-diisopropylethylamine are dissolved in dry dichloromethane, and the molar ratio of the four is 2:1:2:2 or
化合物I或化合物II与化合物IV、缩合剂、N,N-二异丙基乙胺溶于干燥的二氯甲烷中，四者的摩尔比为1:1:2:2或Compound I or compound II and compound IV, condensing agent, N,N-diisopropylethylamine are dissolved in dry dichloromethane, and the molar ratio of the four is 1:1:2:2 or
胆固醇与化合物IV、缩合剂、N,N-二异丙基乙胺溶于干燥的二氯甲烷中，四者的摩尔比为2：1：2：2，溶剂量以其完全溶解为限，在氮气保护下冷却至0℃后在室温下搅拌24小时，之后将反应混合物溶解在二氯甲烷中，依次用5％柠檬酸溶液、水、饱和盐水洗涤，干燥后纯化目的物；Cholesterol, compound IV, condensing agent, and N,N-diisopropylethylamine are dissolved in dry dichloromethane, the molar ratio of the four is 2:1:2:2, and the amount of solvent is limited to its complete dissolution. After cooling to 0°C under the protection of nitrogen, stir at room temperature for 24 hours, then dissolve the reaction mixture in dichloromethane, wash with 5% citric acid solution, water, and saturated brine successively, and purify the target compound after drying;
上述式中，R3选自以下任一个基团：In the above formula, R3 is selected from any of the following groups:
n为1或2；m为3或4。n is 1 or 2; m is 3 or 4.
上述方法中，所述的有机溶剂包括二氯甲烷、三氯甲烷、乙酸乙酯、四氢呋喃、二甲基亚砜、N,N-二甲基甲酰胺、1，4-二氧六环、甲醇、乙醇、乙醚、丙酮、苯、甲苯；In the above method, the organic solvent includes dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, 1,4-dioxane, methanol , ethanol, ether, acetone, benzene, toluene;
上述方法中，每个化学反应均使用碱性催化剂，所述碱性催化剂包括三乙胺、吡啶、4-二甲氨基吡啶，In the above method, each chemical reaction uses a basic catalyst, and the basic catalyst includes triethylamine, pyridine, 4-dimethylaminopyridine,
每一步化学反应完成后，均对产物采用硅胶柱层析进行纯化，硅胶柱层析用的有机溶剂包括乙酸乙酯、正己烷、二氯甲烷、甲醇、乙醇、乙腈按照不同配比混合所得的溶剂体系。After each step of the chemical reaction is completed, the product is purified by silica gel column chromatography. The organic solvent used for silica gel column chromatography includes ethyl acetate, n-hexane, methylene chloride, methanol, ethanol, and acetonitrile mixed according to different proportions. solvent system.
所述的缩合剂为1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、二环己基碳二亚胺、二异丙基碳二亚胺、羰基二咪唑。The condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, diisopropylcarbodiimide, carbonyldiimidazole.
步骤(1)所述的过量酸包括盐酸、磷酸、甲磺酸和/或三氟乙酸。The excess acid described in step (1) includes hydrochloric acid, phosphoric acid, methanesulfonic acid and/or trifluoroacetic acid.
本发明的有益效果在于：本发明所述含有阳离子脂质类化合物具有以下优点：(1)制备工艺简单、产率高；(2)本脂质类分子水溶性高、与质粒DNA结合力强、易形成脂质体；(3)特定比例的α-氨基和δ-氨基，如α-氨基：δ-氨基为2～4：1，使本脂质类化合物具有极高的生物相容性、与市面上现有的转染试剂(如，lipofectamine2000、lipofectamine3000、PEI等)相比较，本脂质类分子的细胞毒性明显低于前者；(4)基因(质粒DNA)转染效率高、由于毒性低，在转染过程中不需要换培养基，有效降低细胞培养及基因转染的成本。The beneficial effect of the present invention is that: the cationic lipid-containing compound of the present invention has the following advantages: (1) the preparation process is simple and the yield is high; (2) the lipid molecule has high water solubility and strong binding force with plasmid DNA , easy to form liposomes; (3) a specific ratio of α-amino and δ-amino, such as α-amino: δ-amino is 2 to 4:1, which makes the lipid compound have extremely high biocompatibility , compared with the existing transfection reagents (such as lipofectamine2000, lipofectamine3000, PEI etc.) on the market, the cytotoxicity of this lipid molecule is obviously lower than the former; Low toxicity, no need to change the medium during the transfection process, effectively reducing the cost of cell culture and gene transfection.
利用本发明的化合物制备得到的基因载体粒径在100nm左右，大小均一，分散性良好；采用凝胶电泳阻滞实验检测脂质基因载体与siRNA的结合能力，结果显示，当N/P比例大于或等于2时，载体能够有效的阻滞质粒的电泳，包裹能力强；本发明的基因载体具有很低的细胞毒性，转染4小时后细胞存活率达到90％，明显高于商品化的脂质体lipofectamine2000；本发明的基因载体与GFP表达质粒混合的复合物，其转染能力强，略高于lipofectamine 2000的转染能力。The particle size of the gene carrier prepared by using the compound of the present invention is about 100nm, uniform in size, and good in dispersion; the binding ability of the lipid gene carrier and siRNA is detected by gel electrophoresis retardation experiment, and the results show that when the N/P ratio is greater than or equal to 2, the carrier can effectively block the electrophoresis of the plasmid, and has a strong wrapping ability; the gene carrier of the present invention has very low cytotoxicity, and the cell survival rate reaches 90% after 4 hours of transfection, which is significantly higher than that of commercially available liposomes. Plastid lipofectamine2000; the compound of the gene carrier and GFP expression plasmid of the present invention has a strong transfection ability, which is slightly higher than that of lipofectamine 2000.
附图说明Description of drawings
图1为由实施例3所得脂类分子化合物制得的基因载体形成的脂质体粒度分布。Fig. 1 is the particle size distribution of liposomes formed by the gene carrier prepared from the lipid molecular compound obtained in Example 3.
图2为使用实施例4所得脂类分子化合物制得的基因载体形成的脂质体，在HEK293细胞和HepG2细胞上转染pcDNA3-eGFP质粒，24小时后用荧光显微镜观察绿色荧光蛋白表达量。对照为lipofectamine2000试剂。Fig. 2 is the liposome formed by the gene carrier prepared by using the lipid molecular compound obtained in Example 4, transfected with pcDNA3-eGFP plasmid on HEK293 cells and HepG2 cells, and observing the expression of green fluorescent protein with a fluorescence microscope after 24 hours. The control was lipofectamine2000 reagent.
图3为实施例3中所得脂类分子化合物制得的基因载体的质谱分析结果。Fig. 3 is the mass spectrometric analysis result of the gene carrier prepared from the lipid molecular compound obtained in Example 3.
图4为实施例3所得脂类分子化合物制得的基因载体与质粒DNA结合的凝胶电泳图；图中0、1、2、3、4分别代表脂质体上的氨基数(N)与DNA分子上的磷酸基数(P)的摩尔比例。Fig. 4 is the gel electrophoresis figure that the gene carrier that the obtained lipid molecular compound of embodiment 3 makes is combined with plasmid DNA; Among the figure, 0, 1, 2, 3, 4 represent the amino number (N) and the number of amino groups (N) on the liposome respectively The molar ratio of phosphate groups (P) on a DNA molecule.
图5为实施例4所得脂类分子化合物制得的基因载体的细胞毒性试验结果。Fig. 5 is the cytotoxicity test result of the gene vector prepared from the lipid molecular compound obtained in Example 4.
具体实施方式Detailed ways
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.
若未特别指明，实施例中所用的化学试剂均为常规市售试剂，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.
除非另有说明，上下文中百分比为重量百分比，所有的温度以摄氏度给出。Unless otherwise stated, percentages in this context are by weight and all temperatures are given in degrees Celsius.
实施例1原料的合成The synthesis of embodiment 1 raw material
化合物I(谷氨酸的饱和/不饱和脂肪烃酰胺)的合成：将饱和/不饱和烃基氨或醇、Boc-谷氨酸-OH溶于干燥的有机溶剂中，在氮气保护下加入缩合剂，在0～35℃下搅拌12～24h后，蒸馏除去有机溶剂，加过量酸的混合液，在室温搅拌1～2h后蒸馏除去溶剂，用硅胶柱层析法分离纯化得到中间体谷氨酸的饱和/不饱和烃基取代酯或酰胺，产率为70％。Synthesis of compound I (saturated/unsaturated aliphatic hydrocarbon amide of glutamic acid): dissolve saturated/unsaturated hydrocarbon-based ammonia or alcohol, Boc-glutamic acid-OH in a dry organic solvent, and add a condensing agent under nitrogen protection , after stirring at 0-35°C for 12-24 hours, distill off the organic solvent, add excess acid mixture, stir at room temperature for 1-2 hours, distill off the solvent, and use silica gel column chromatography to separate and purify the intermediate glutamic acid Saturated/unsaturated hydrocarbyl substituted esters or amides with a yield of 70%.
化合物II(天冬氨酸的饱和/不饱和脂肪烃酰胺)的合成：将饱和/不饱和烃基氨或醇、Boc-天冬氨酸-OH溶于干燥的有机溶剂中，在氮气保护下加入缩合剂，在0～35℃下搅拌12～24h后，蒸馏除去有机溶剂，加过量酸的混合液，在室温搅拌1～2h后蒸馏除去溶剂，用硅胶柱层析法分离纯化得到中间体谷氨酸的饱和/不饱和烃基取代酯或酰胺，即化合物I，产率为75％。Synthesis of compound II (saturated/unsaturated aliphatic hydrocarbon amide of aspartic acid): dissolve saturated/unsaturated hydrocarbon-based ammonia or alcohol, Boc-aspartic acid-OH in a dry organic solvent, add under nitrogen protection Condensing agent, stir at 0-35°C for 12-24 hours, distill off the organic solvent, add excess acid mixture, stir at room temperature for 1-2 hours, distill off the solvent, and use silica gel column chromatography to separate and purify to obtain the intermediate valley Saturated/unsaturated hydrocarbyl substituted ester or amide of amino acid, ie compound I, with a yield of 75%.
实施例2原料的合成The synthesis of embodiment 2 raw materials
化合物III(Boc保护的仿肽类分子)的合成：Synthesis of Compound III (Boc-protected peptidomimetics):
第一步：将Boc-鸟氨酸、Boc-鸟氨酸(Boc)-OSu在碱性条件下有机溶剂中在0～35℃下搅拌24～36h，蒸馏除去有机溶剂后用硅胶柱层析法分离纯化得到中间体Boc-鸟氨酸(Boc)-鸟氨酸(Boc)-COOH，所述的Boc是碳酸二叔丁酯。产率：60％。The first step: Stir Boc-ornithine and Boc-ornithine (Boc)-OSu in an organic solvent under alkaline conditions at 0-35°C for 24-36 hours, distill off the organic solvent and use silica gel column chromatography The intermediate Boc-ornithine (Boc)-ornithine (Boc)-COOH is obtained by separation and purification by the method, and the Boc is di-tert-butyl carbonate. Yield: 60%.
第二步：将由第一步得到的中间体、N-羟基琥珀酰亚胺、缩合剂在有机溶剂中搅拌12h，再加入Boc-鸟氨酸，在0～35℃下继续搅拌12～24h后蒸馏除去有机溶剂后用硅胶柱层析法分离纯化得到化合物II，Boc-鸟氨酸(Boc)-鸟氨酸(Boc)-鸟氨酸(Boc)-COOH。所述的Boc是碳酸二叔丁酯。产率：65％。The second step: Stir the intermediate, N-hydroxysuccinimide, and condensing agent obtained in the first step in an organic solvent for 12 hours, then add Boc-ornithine, and continue stirring at 0-35°C for 12-24 hours After the organic solvent was distilled off, the compound II, Boc-Ornithine (Boc)-Ornithine (Boc)-Ornithine (Boc)-COOH, was obtained by separation and purification by silica gel column chromatography. The Boc is di-tert-butyl carbonate. Yield: 65%.
化合物IV(Boc保护的仿肽类分子)的合成：Synthesis of Compound IV (Boc-protected peptidomimetics):
第一步：将Boc-赖氨酸、Boc-赖氨酸(Boc)-OSu在碱性条件下有机溶剂中在0～35℃下搅拌24～36h，蒸馏除去有机溶剂后用硅胶柱层析法分离纯化得到中间体Boc-赖氨酸(Boc)-赖氨酸(Boc)-COOH，所述的Boc是碳酸二叔丁酯。产率：60％。Step 1: Stir Boc-lysine and Boc-lysine (Boc)-OSu in an organic solvent under alkaline conditions at 0-35°C for 24-36 hours, distill off the organic solvent and use silica gel column chromatography The intermediate Boc-lysine (Boc)-lysine (Boc)-COOH is obtained by separation and purification by the method, and the Boc is di-tert-butyl carbonate. Yield: 60%.
第二步：将由第一步得到的中间体、N-羟基琥珀酰亚胺、缩合剂在有机溶剂中搅拌12h，再加入Boc-赖氨酸，在0～35℃下继续搅拌12～24h后蒸馏除去有机溶剂后用硅胶柱层析法分离纯化得到化合物IV，Boc-赖氨酸(Boc)-赖氨酸(Boc)-赖氨酸(Boc)-COOH。所述的Boc是碳酸二叔丁酯。产率：68％。The second step: Stir the intermediate, N-hydroxysuccinimide, and condensing agent obtained in the first step in an organic solvent for 12 hours, then add Boc-lysine, and continue stirring at 0-35°C for 12-24 hours After the organic solvent was distilled off, the compound IV, Boc-lysine (Boc)-lysine (Boc)-lysine (Boc)-COOH, was obtained by separation and purification by silica gel column chromatography. The Boc is di-tert-butyl carbonate. Yield: 68%.
本发明中所述的一种脂质阳离子有机功能分子的合成步骤中所述的有机溶剂包括二氯甲烷、三氯甲烷、乙酸乙酯、四氢呋喃、二甲基亚砜、N,N-二甲基甲酰胺、1，4-二氧六环、甲醇、乙醇、乙醚、丙酮、苯、甲苯；每个反应均使用碱性催化剂，碱性催化剂包括三乙胺、吡啶、4-二甲氨基吡啶，所述柱层析用的有机溶剂包括乙酸乙酯、正己烷、二氯甲烷、甲醇、乙醇、乙醚、乙腈按照不同配比混合所得的溶剂体系。The organic solvent described in the synthesis step of a lipid cationic organic functional molecule described in the present invention includes dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethyl methyl formamide, 1,4-dioxane, methanol, ethanol, ether, acetone, benzene, toluene; each reaction uses a basic catalyst, including triethylamine, pyridine, 4-dimethylaminopyridine , the organic solvent used in the column chromatography includes a solvent system obtained by mixing ethyl acetate, n-hexane, dichloromethane, methanol, ethanol, ether, and acetonitrile according to different proportions.
本发明中所述的一种脂质阳离子有机功能分子的合成步骤中所述的缩合剂为1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDCI)、二环己基碳二亚胺(DCC)、二异丙基碳二亚胺(DIC)、羰基二咪唑(CDI)。The condensing agent described in the synthesis step of a lipid cationic organic functional molecule described in the present invention is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), Dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), carbonyldiimidazole (CDI).
本发明中所述的一种脂质阳离子有机功能分子的合成步骤中，所述的过量酸包括盐酸、磷酸、甲磺酸、三氟乙酸。In the synthesis step of a lipid cationic organic functional molecule described in the present invention, the excess acid includes hydrochloric acid, phosphoric acid, methanesulfonic acid, and trifluoroacetic acid.
本发明中所述的一种脂质阳离子有机功能分子的合成步骤中所述的反应温度优选但不限0～35℃，特别优选25～35℃。The reaction temperature described in the synthesis step of a lipid cationic organic functional molecule in the present invention is preferably but not limited to 0-35°C, particularly preferably 25-35°C.
实施例3本发明化合物的制备(1)Embodiment 3 The preparation of compound of the present invention (1)
原料包括化合物I(谷氨酸脂肪烷烃酰胺分子)、化合物III(氨基Boc保护的短肽类分子)、缩合剂(1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐EDCI)、碱(N,N-二异丙基乙胺DIPEA)溶于干燥的二氯甲烷中，化合物I：化合物III：缩合剂：碱(摩尔比)＝1：1：2：2，溶剂量以化合物I、化合物III、缩合剂、碱完全溶解为限。Raw materials include compound I (glutamic acid fatty alkane amide molecule), compound III (amino Boc-protected short peptide molecule), condensation agent (1-ethyl-(3-dimethylaminopropyl) carbodiimide salt Acid EDCI), base (N,N-diisopropylethylamine DIPEA) dissolved in dry dichloromethane, compound I: compound III: condensing agent: base (molar ratio) = 1:1:2:2 , The amount of solvent is limited to the complete dissolution of compound I, compound III, condensing agent and base.
在氮气保护下用冰浴冷却至0℃后在25℃搅拌24小时。反应结束后，将反应混合物溶解在二氯甲烷中，依次用5％柠檬酸溶液、水、饱和盐水洗涤3次，用无水硫酸钠干燥后用硅胶柱层析方法(洗脱液：二氯甲烷/甲醇＝15/1，v/v)纯化目的物，得到目的物2.13g产物，产率82％。Under the protection of nitrogen, the mixture was cooled to 0°C with an ice bath and then stirred at 25°C for 24 hours. After the reaction was over, the reaction mixture was dissolved in dichloromethane, washed 3 times with 5% citric acid solution, water, and saturated brine successively, dried with anhydrous sodium sulfate, and then purified by silica gel column chromatography (eluent: dichloromethane Methane/methanol=15/1, v/v) Purify the target object to obtain 2.13 g of the target product with a yield of 82%.
产物表征参数为：1HNMR(DMSO-d6,ppm):0.838-0.865(t,6H,-CH3),1.235-1.562(m,48H,-CH2-,-CONHCH2CH2-),1.576-1.707(m,16H,-NHCOCH2-,-NHCOCH2CH2,-NHCOCH(NH2)CH2-,-NHCOCH(NH2)CH2CH2-),1.938-2.098(t,8H,-CH2CH＝CHCH2-),2.805(m,2H,NH2CH2-),2.993-3.006(m,2H,-CONHCH2-),3.119-3.170(m,6H,-CH2(NH)CHCONHCH2-,NH2CONHCH2-),3.753(m,3H,-NHCOCH(NH2)-),4.271(m,1H,-NHCOCH(NH)CH2-),5.311-5.352(m,4H,-CH＝CH-),7.809-8.612(m,14H,CONH-,-NH2).MS(ESI)m/z 988.76(M)+。(见图3)表征数据说明结构正确。Product characterization parameters are:1 HNMR(DMSO-d6,ppm):0.838-0.865(t,6H,-CH3 ),1.235-1.562(m,48H,-CH2 -,-CONHCH2 CH2 -),1.576 -1.707(m,16H,-NHCOCH2 -,-NHCOCH2 CH2 ,-NHCOCH(NH2 )CH2 -,-NHCOCH(NH2 )CH2 CH2 -),1.938-2.098(t,8H,- CH2 CH=CHCH2 -),2.805(m,2H,NH2 CH2 -),2.993-3.006(m,2H,-CONHCH2 -),3.119-3.170(m,6H,-CH2 (NH) CHCONHCH2 -,NH2 CONHCH2 -),3.753(m,3H,-NHCOCH(NH2 )-),4.271(m,1H,-NHCOCH(NH)CH2 -),5.311-5.352(m,4H, -CH=CH-), 7.809-8.612 (m, 14H, CONH-, -NH2 ). MS (ESI) m/z 988.76 (M)+ . (See Figure 3) The characterization data indicated that the structure was correct.
实施例4本发明化合物的制备(2)The preparation of embodiment 4 compounds of the present invention (2)
原料包括化合物II(天冬氨酸脂肪烷烃酰胺分子)、化合物III(氨基Boc保护的短肽类分子)、缩合剂(1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐EDCI)、碱(N,N-二异丙基乙胺DIPEA)溶于干燥的二氯甲烷中，化合物II：化合物III：缩合剂：碱(摩尔比)＝1：1：2：2，溶剂量以化合物II、化合物III、缩合剂、碱完全溶解为限。Raw materials include compound II (aspartic acid fatty alkane amide molecule), compound III (short peptide molecule protected by amino Boc), condensation agent (1-ethyl-(3-dimethylaminopropyl) carbodiimide Hydrochloride EDCI), base (N,N-diisopropylethylamine DIPEA) dissolved in dry dichloromethane, compound II: compound III: condensing agent: base (molar ratio) = 1:1:2: 2. The amount of solvent is limited to the complete dissolution of compound II, compound III, condensing agent and alkali.
在氮气保护下用冰浴冷却至0℃后在25℃搅拌24小时。反应结束后，将反应混合物溶解在二氯甲烷中，依次用5％柠檬酸溶液、水、饱和盐水洗涤3次，用无水硫酸钠干燥后用硅胶柱层析方法(洗脱液：二氯甲烷/甲醇＝15/1，v/v)纯化目的物，产率85％。Under the protection of nitrogen, the mixture was cooled to 0°C with an ice bath and then stirred at 25°C for 24 hours. After the reaction was over, the reaction mixture was dissolved in dichloromethane, washed 3 times with 5% citric acid solution, water, and saturated brine successively, dried with anhydrous sodium sulfate, and then purified by silica gel column chromatography (eluent: dichloromethane Methane/methanol=15/1, v/v) to purify the target product with a yield of 85%.
产物表征参数为：1HNMR(DMSO-d6,ppm):0.838-0.865(t,6H,-CH3),1.235-1.562(m,48H,-CH2-,-CONHCH2CH2-),1.576-1.707(m,16H,-NHCOCH2-,-NHCOCH2CH2,-NHCOCH(NH2)CH2-,-NHCOCH(NH2)CH2CH2-),1.938-2.098(t,8H,-CH2CH＝CHCH2-),2.805(m,2H,NH2CH2-),2.993-3.006(m,2H,-CONHCH2-),3.119-3.170(m,6H,-CH2(NH)CHCONHCH2-,NH2CONHCH2-),3.753(m,3H,-NHCOCH(NH2)-),4.271(m,1H,-NHCOCH(NH)CH2-),5.311-5.352(m,4H,-CH＝CH-),7.809-8.612(m,14H,CONH-,-NH2).MS(ESI)m/z 974.7(M)+。(见图3)表征数据说明结构正确。Product characterization parameters are:1 HNMR(DMSO-d6,ppm):0.838-0.865(t,6H,-CH3 ),1.235-1.562(m,48H,-CH2 -,-CONHCH2 CH2 -),1.576 -1.707(m,16H,-NHCOCH2 -,-NHCOCH2 CH2 ,-NHCOCH(NH2 )CH2 -,-NHCOCH(NH2 )CH2 CH2 -),1.938-2.098(t,8H,- CH2 CH=CHCH2 -),2.805(m,2H,NH2 CH2 -),2.993-3.006(m,2H,-CONHCH2 -),3.119-3.170(m,6H,-CH2 (NH) CHCONHCH2 -,NH2 CONHCH2 -),3.753(m,3H,-NHCOCH(NH2 )-),4.271(m,1H,-NHCOCH(NH)CH2 -),5.311-5.352(m,4H, -CH=CH-), 7.809-8.612 (m, 14H, CONH-, -NH2 ). MS (ESI) m/z 974.7 (M)+ . (See Figure 3) The characterization data indicated that the structure was correct.
实施例5本发明化合物的制备(3)Embodiment 5 The preparation of compound of the present invention (3)
原料包括化合物III(氨基Boc保护的短肽类分子)、胆固醇、缩合剂(1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐EDCI)、碱(N,N-二异丙基乙胺DIPEA)溶于干燥的二氯甲烷中，化合物III：胆固醇：缩合剂：碱(摩尔比)＝1：2：2：2，溶剂量以化合物III、胆固醇、缩合剂、碱完全溶解为限。Raw materials include compound III (short peptide molecule protected by amino Boc), cholesterol, condensation agent (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride EDCI), base (N,N -Diisopropylethylamine (DIPEA) is dissolved in dry dichloromethane, compound III: cholesterol: condensing agent: alkali (molar ratio)=1:2:2:2, the amount of solvent is compound III, cholesterol, condensing agent , Alkaline complete dissolution is limited.
将化合物III、胆固醇、缩合剂、碱按比例称取，在氮气保护下用冰浴冷却至0℃后在25℃搅拌24小时。反应结束后，将反应混合物溶解在二氯甲烷中，依次用5％柠檬酸溶液、水、饱和盐水洗涤3次，用无水硫酸钠干燥后用硅胶柱层析方法(洗脱液：二氯甲烷/甲醇＝15/1，v/v)纯化目的物，产率79％。对目的物进行氢谱质谱验证，其表征参数说明该化合物结构正确。Compound III, cholesterol, condensing agent, and base were weighed in proportion, cooled to 0° C. in an ice bath under nitrogen protection, and then stirred at 25° C. for 24 hours. After the reaction was over, the reaction mixture was dissolved in dichloromethane, washed 3 times with 5% citric acid solution, water, and saturated brine successively, dried with anhydrous sodium sulfate, and then purified by silica gel column chromatography (eluent: dichloromethane Methane/methanol=15/1, v/v) to purify the target product with a yield of 79%. The target compound was verified by hydrogen spectrum mass spectrometry, and its characterization parameters indicated that the structure of the compound was correct.
实施例6含本发明化合物的脂质体转基因载体的制备与生物学活性验证Example 6 Preparation and Biological Activity Verification of Liposome Transgenic Vectors Containing Compounds of the Present Invention
分别将由实施例3、实施例4、实施例5得到的脂质分子化合物与DOPE或DOPC按3～1：1～3的摩尔比例混合，溶解在氯仿中，减压蒸馏移去氯仿后真空干燥过夜；加入pH7.2的PBS混匀后，在室温放置1小时；用Avanti Mini-Extruder脂质体挤出器(含孔径100纳米的膜)反复挤出(20到50次)，制备得到脂质体基因载体，在4℃冰箱内保存。对制备得到的新型脂质体转基因载体，对实施例3-5的化合物制得的基因载体其进行下述几个方面的检测：Mix the lipid molecule compounds obtained in Example 3, Example 4, and Example 5 with DOPE or DOPC in a molar ratio of 3 to 1:1 to 3, dissolve them in chloroform, remove the chloroform by distillation under reduced pressure, and then dry in vacuo Overnight; after adding PBS with pH 7.2 and mixing, leave it at room temperature for 1 hour; repeatedly extrude (20 to 50 times) with an Avanti Mini-Extruder liposome extruder (with a membrane with a pore size of 100 nm) to prepare a liposome Plastid gene vectors, stored in a refrigerator at 4°C. To the novel liposome transgene carrier prepared, the gene carrier that the compound of embodiment 3-5 makes it carries out the detection of following several aspects:
(1)利用激光粒度仪检测载体的大小、粒径分布情况，图1显示结果，实施例3的化合物制得的基因载体粒径在100 nm到150nm左右，大小均一，分散性良好。实施例4、5得到的化合物制得的基因载体的效果同此。(1) Use a laser particle size analyzer to detect the size and particle size distribution of the carrier. Figure 1 shows the results. The particle size of the gene carrier prepared by the compound of Example 3 is about 100 nm to 150 nm, with uniform size and good dispersion. The effect of the gene carrier prepared by the compound obtained in Examples 4 and 5 is the same.
(2)采用凝胶电泳阻滞实验检测脂质基因载体与pcDNA3-eGFP质粒的结合能力，结果显示，当氨基：磷酸基团比例(即，N/P比例)大于或等于2时，载体能够有效的阻滞质粒的电泳，说明其结合和包裹DNA的能力强(见图4)。实施例4、5得到的化合物制得的基因载体的效果同此。(2) Gel electrophoresis retardation test was used to detect the binding ability of the lipid gene carrier and the pcDNA3-eGFP plasmid. The results showed that when the amino group ratio (that is, the N/P ratio) was greater than or equal to 2, the carrier could The electrophoresis of the effectively retarded plasmid shows that it has a strong ability to bind and wrap DNA (see Figure 4). The effect of the gene carrier prepared by the compound obtained in Examples 4 and 5 is the same.
(3)细胞转染效率的测定：(3) Determination of cell transfection efficiency:
HepG2细胞、HEK293细胞的培养：在含有10％的胎牛血清的培养液中，在37℃，5％CO2条件下培养24小时。Cultivation of HepG2 cells and HEK293 cells: in a culture solution containing 10% fetal bovine serum, cultured at 37° C. and 5% CO2 for 24 hours.
转染前一天，按每孔2.5×105细胞密度接种于12孔培养板，在5％CO2、温度为37℃的细胞培养箱中继续培养到细胞密度达到70％左右；更换含有10％小牛血清的新鲜培养液(1.0 ml/孔)；将pcDNA3-eGFP质粒(1.0ug在100 ul Opti-MEM培养液中)和5-7微升的脂质体转染试剂(由实施例4所得)(在100ul Opti-MEM培养液中)，按1：1体积比例混合两种溶液，在室温放置30分钟后，加入到细胞上面，继续培养48小时；The day before transfection, inoculate 2.5×105 cells per well in a 12-well culture plate, and continue culturing in a cell incubator with 5% CO2 and a temperature of 37°C until the cell density reaches about 70%; replace with 10% Fresh culture fluid (1.0 ml/hole) of calf serum; pcDNA3-eGFP plasmid (1.0ug in 100 ul Opti-MEM culture fluid) and the liposome transfection reagent of 5-7 microliters (from embodiment 4 (obtained) (in 100ul Opti-MEM culture medium), mix the two solutions according to the volume ratio of 1:1, leave it at room temperature for 30 minutes, add it to the top of the cells, and continue to cultivate for 48 hours;
转染效率的测定：在荧光倒置显微镜下观察并取照相，如图2所示。结果显示采用本发明实施例4得到的化合物制得的基因载体与GFP表达质粒混合的复合物，其转染能力强，与商品化lipofectamine2000转染试剂相同或略高。实施例3、5的化合物制得的基因载体效果与此相同。Determination of transfection efficiency: observe and take pictures under a fluorescent inverted microscope, as shown in FIG. 2 . The results show that the complex of the gene carrier and the GFP expression plasmid prepared by the compound obtained in Example 4 of the present invention has a strong transfection ability, which is the same as or slightly higher than that of the commercial lipofectamine2000 transfection reagent. The effect of the gene vector prepared by the compounds of Examples 3 and 5 is the same.
(4)细胞存活率检测：(4) Detection of cell viability:
转染前一天，按每孔1.0×104细胞密度接种于96孔培养板，在5％CO2、温度为37℃的细胞培养箱中继续培养到细胞密度达到70％左右；更换含有10％小牛血清的新鲜培养液(1.0 ml/孔)；将pcDNA-GFP质粒(1.0ug在100 ul Opti-MEM培养液中)和脂质转染试剂(实施例4得到的化合物制得的基因载体，5～7ul在100 ulOpti-MEM培养液中)，混合两种溶液，在室温放置30分钟后，取1.0ul，加入到细胞上面，继续培养48小时后，采用MTT方法，测定细胞存活率，其计算公式为：The day before transfection, inoculate 1.0×104 cells per well in a 96-well culture plate, and continue culturing in a cell incubator with 5% CO2 and a temperature of 37°C until the cell density reaches about 70%; replace with 10% Fresh nutrient solution (1.0 ml/well) of calf serum; pcDNA-GFP plasmid (1.0ug in 100 ul Opti-MEM nutrient solution) and lipofection reagent (the gene carrier that the compound that embodiment 4 obtains makes , 5 ~ 7ul in 100ul Opti-MEM culture medium), mix the two solutions, place at room temperature for 30 minutes, take 1.0ul, add to the top of the cells, continue to culture for 48 hours, use the MTT method to measure the cell survival rate, Its calculation formula is:
[细胞存活率(％)＝(Asample/Acontrol)×100，其中，[cell survival rate (%)=(Asample/Acontrol)×100, wherein,
Asample是被转染细胞孔的吸收，Acontrol是没有经过转染的细胞孔的吸收，每组实验重复6次，结果如图5所示。结果显示，HEK293细胞在转染4小时后细胞存活率达到95％，明显高于lipofectamine2000(细胞存活率为70％)和lipofectamine 3000(细胞存活率为85％)，实施例3、5的化合物制得的基因载体效果与此相同，细胞转染4小时候细胞存活率达到95％，可见本发明的基因载体具有很低的细胞毒性。Asample is the absorption of transfected cell wells, and Acontrol is the absorption of non-transfected cell wells. Each experiment was repeated 6 times, and the results are shown in Figure 5. The results showed that HEK293 cells reached 95% cell viability 4 hours after transfection, which was significantly higher than lipofectamine 2000 (cell viability 70%) and lipofectamine 3000 (cell viability 85%). The effect of the obtained gene carrier is the same, and the cell survival rate reaches 95% after 4 hours of cell transfection, which shows that the gene carrier of the present invention has very low cytotoxicity.
以上的实施例仅仅是对本发明的优选实施方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通工程技术人员对本发明的技术方案作出的各种变型和改进，均应落入本发明的权利要求书确定的保护范围内。The above embodiments are only descriptions of preferred implementations of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, ordinary engineers and technicians in the field may make various modifications to the technical solutions of the present invention. and improvements, all should fall within the scope of protection determined by the claims of the present invention.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510228936.4ACN106188230B (en) | 2015-05-07 | 2015-05-07 | A kind of cationic lipid compound and its preparation method and application |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510228936.4ACN106188230B (en) | 2015-05-07 | 2015-05-07 | A kind of cationic lipid compound and its preparation method and application |
| Publication Number | Publication Date |
|---|---|
| CN106188230A CN106188230A (en) | 2016-12-07 |
| CN106188230Btrue CN106188230B (en) | 2019-10-25 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510228936.4AExpired - Fee RelatedCN106188230B (en) | 2015-05-07 | 2015-05-07 | A kind of cationic lipid compound and its preparation method and application |
| Country | Link |
|---|---|
| CN (1) | CN106188230B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108912201B (en)* | 2018-08-07 | 2020-12-15 | 江南大学 | A kind of preparation method of ornithine phytosterol ester hydrochloride |
| CN113292616B (en)* | 2021-05-20 | 2023-05-23 | 内蒙古大学 | Monosaccharide ligand functionalized cationic lipid compound and preparation method and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1177342A (en)* | 1995-02-21 | 1998-03-25 | ImaRx药物公司 | Novel cationic lipids and the use thereof |
| CN1277024A (en)* | 1999-06-11 | 2000-12-20 | 克林诺斯生物药品工业有限公司 | Use of complexes of cationic liposomes and polydeoxyribonucleotides as medicaments |
| CN102911252A (en)* | 2012-10-25 | 2013-02-06 | 四川大学 | Cationic lipid containing peptide dendrimer, transgenic carrier and preparation method and application of transgenic carrier |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1177342A (en)* | 1995-02-21 | 1998-03-25 | ImaRx药物公司 | Novel cationic lipids and the use thereof |
| CN1277024A (en)* | 1999-06-11 | 2000-12-20 | 克林诺斯生物药品工业有限公司 | Use of complexes of cationic liposomes and polydeoxyribonucleotides as medicaments |
| CN102911252A (en)* | 2012-10-25 | 2013-02-06 | 四川大学 | Cationic lipid containing peptide dendrimer, transgenic carrier and preparation method and application of transgenic carrier |
| Publication number | Publication date |
|---|---|
| CN106188230A (en) | 2016-12-07 |
| Publication | Publication Date | Title |
|---|---|---|
| EP2635265B1 (en) | Novel low molecular weight cyclic amine containing cationic lipids for oligonucleotide delivery | |
| CN111087317B (en) | Unsaturated cationic lipid derivative, preparation method and application in plasmid delivery system | |
| JP4467084B2 (en) | Compounds for introducing nucleic acids into cells, their production and their use | |
| EP2647713B1 (en) | Modified single-strand polynucleotide | |
| EP0968227A1 (en) | Dimeric cationic lipids on dicystine basis | |
| CA3215389C (en) | Ionizable lipids and compositions for nucleic acid delivery | |
| WO2023109243A1 (en) | Ionizable lipid compound and nucleic acid in vitro cell transfection reagent | |
| CN113121381B (en) | Ceramide compound, cationic liposome thereof, preparation method and application | |
| CN116143855B (en) | Vinyl phosphonic acid modified mRNA cap analogue and preparation method and application thereof | |
| CN102911252A (en) | Cationic lipid containing peptide dendrimer, transgenic carrier and preparation method and application of transgenic carrier | |
| EP3539964B1 (en) | Base acetamide glycerin ether molecule, chemical synthesis method therefor, and applications thereof in field of gene therapy | |
| CN101870719B (en) | Organic functional compound having disulfide chemical bond and steroids skeleton, preparation method thereof and use thereof | |
| WO2003057164A2 (en) | Compounds for delivering substances into cells | |
| CN106188230B (en) | A kind of cationic lipid compound and its preparation method and application | |
| US10294199B2 (en) | Propyl cationic peptide lipids, synthesis method thereof, and application thereof | |
| US10597678B2 (en) | Bis-alkoxyl amide alkyl cationic peptide lipids, synthesis method thereof, and application thereof | |
| CN102161688A (en) | Cholesterol-structure-fragment-containing biocompatible synthesis liposomes as well as preparation method and application thereof | |
| CN101475503B (en) | Novel cation lipoid, preparation and use thereof | |
| EP1187806B1 (en) | Spermine:peptide-based surfactant compounds | |
| CN106188223B (en) | A compound containing dipeptide lipid cation and its preparation method and application | |
| CN103772469A (en) | Synthetic cation lipid with zoosterol and natural arginine structural fragment and synthesis method and application thereof | |
| CN114349811A (en) | Cationic cholesterol derivative, nano-composite, preparation method and application thereof | |
| CN101732730B (en) | Cation lipid containing imidazolium salt and macrocyclic polyamines, transgenetic vector and method for preparing same | |
| CN108395411B (en) | Dendrimer-like cationic lipid, transgenic vector and preparation method thereof | |
| CN113292616B (en) | Monosaccharide ligand functionalized cationic lipid compound and preparation method and application thereof |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | Granted publication date:20191025 | |
| CF01 | Termination of patent right due to non-payment of annual fee |