技术领域technical field
本发明属于基因治疗技术领域，具体涉及一种3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸的两亲性衍生物及其用途。The invention belongs to the technical field of gene therapy, and in particular relates to an amphiphilic derivative of 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid and its application.
背景技术Background technique
基因治疗(gene therapy)是指将外源正常基因导入靶细胞，以纠正或补偿因基因缺陷和异常引起的疾病，以达到治疗目的。在过去的二十多年间，基因治疗在很多疾病治疗领域将研究从临床前推向了临床，对于由基因异常引起的，医药界至今难以解决的疾病如肿瘤等具有无可替代的优势。常见的基因药物有质粒DNA(plasmid DNA，pDNA)、反义寡核苷酸(antisense ODN)、小干扰RNA(siRNA)和小发卡RNA(shRNA)。具有RNAi干扰效应，能特异性沉默目的基因的siRNA是目前基因治疗研究的重点。Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate diseases caused by gene defects and abnormalities, so as to achieve therapeutic purposes. In the past two decades, gene therapy has pushed the research from preclinical to clinical in many disease treatment fields, and has irreplaceable advantages for diseases caused by gene abnormalities that are difficult to solve in the medical field, such as tumors. Common gene medicines include plasmid DNA (plasmid DNA, pDNA), antisense oligonucleotide (antisense ODN), small interfering RNA (siRNA) and small hairpin RNA (shRNA). siRNA that has RNAi interference effect and can specifically silence the target gene is the focus of current gene therapy research.
siRNA(Small interfering RNA)，又称为小干扰RNA，是长度20到25个核苷酸的双链RNA。通过与之序列互补的mRNA相结合，促使mRNA降解，介导转录水平的基因表达抑制，从而诱使细胞表现出特定基因缺失的表型。siRNA的调控机制是通过互补配对来对相应靶位基因的表达进行沉默，因此具有高度的特异性。所以将其作为治疗药物，具有广阔的发展前景，对于恶性肿瘤、HIV等一系列病症的治疗具有重大意义。siRNA (Small interfering RNA), also known as small interfering RNA, is a double-stranded RNA with a length of 20 to 25 nucleotides. By combining with the complementary mRNA, it promotes the degradation of mRNA and mediates the inhibition of gene expression at the transcriptional level, thereby inducing cells to exhibit a phenotype of specific gene deletion. The regulatory mechanism of siRNA is to silence the expression of the corresponding target gene through complementary pairing, so it has a high degree of specificity. Therefore, it has broad development prospects as a therapeutic drug, and is of great significance for the treatment of a series of diseases such as malignant tumors and HIV.
基因治疗的关键是将基因药物体内输送到靶细胞，使其发挥作用。然而，将外源基因引入体内，其会被体内的核酸酶降解，在未进入靶细胞之前，便被降解成小分子核苷酸，从而失去治疗作用。因此，实现基因治疗的关键是高效、安全的基因递送系统。The key to gene therapy is to deliver gene medicine to target cells in vivo to make it work. However, when exogenous genes are introduced into the body, they will be degraded by nucleases in the body, and before they enter the target cells, they will be degraded into small molecule nucleotides, thus losing their therapeutic effect. Therefore, the key to realizing gene therapy is an efficient and safe gene delivery system.
基因载体在运送基因的时候要经历多个复杂的过程：通过血液循环到达靶细胞，细胞摄取，内涵体的逃逸，胞内运动，载体释放基因物质。其主要障碍主要是复杂血液环境的细胞外障碍和溶酶体酶降解的细胞内障碍。因此寻找良好的基因载体，使得靶基因到达靶点发挥效用，是基因载体研究者亟待解决的问题。The gene carrier has to go through multiple complex processes when transporting the gene: reaching the target cell through blood circulation, cell uptake, escape of endosomes, intracellular movement, and release of gene material by the carrier. Its main barriers are mainly extracellular barriers in the complex blood environment and intracellular barriers in the degradation of lysosomal enzymes. Therefore, it is an urgent problem for gene carrier researchers to find a good gene carrier to make the target gene reach the target site and play a role.
目前，在基因输送载体系统方面主要分为两大类：一是病毒载体系统；二是非病毒载体系统。病毒载体是一种天然的载体资源，病毒基因组结构简单，转染效率高，靶细胞特异性强，但其导向性差、携带能力低、免疫原性和潜在致瘤性的局限性，使其难以达到临床应用的要求。因此多样性、无免疫原性及易于控制生产的非病毒载体系统近年来备受关注，并在很多治疗领域有所应用。常用的非病毒载体系统主要是脂质(cationic lipids)载体。Currently, gene delivery vector systems are mainly divided into two categories: one is viral vector system; the other is non-viral vector system. Viral vector is a natural carrier resource. Viral genome has simple structure, high transfection efficiency and strong target cell specificity. However, its poor orientation, low carrying capacity, limitations of immunogenicity and potential tumorigenicity make it difficult to Meet the requirements of clinical application. Therefore, the non-viral vector system with diversity, non-immunogenicity and easy-to-control production has attracted much attention in recent years and has been applied in many therapeutic fields. Commonly used non-viral vector systems are mainly lipid (cationic lipids) vectors.
阳离子脂质的正电荷通过静电作用与带负电的基因药物结合，从而将基因物质浓缩包装成较小粒径的粒子。带正电的阳离子脂质体可以将基因药物siRNA浓缩装载成粒径较小的纳米粒子。阳离子脂质体在基因输送载体制备和细胞转染方面显示出了其他类型脂质体无法比拟的优势。复合物较小的粒径降低了被体内巨嗜细胞识别、吞噬、清除的机会，提高了药物的体内生物利用度。同时，针对于肿瘤组织，复合物较小粒径更容易利用渗透和滞留效应从血管内皮细胞间隙透过进入肿瘤实质，增加在肿瘤组织的药物聚集。在转染方面，由于细胞表面略微带有负电，带正电的脂质体更容易吸附到细胞表面，通过内吞等机制进入细胞，大大增加了脂质体的转染能力。The positive charge of the cationic lipid is combined with the negatively charged gene drug through electrostatic interaction, so that the gene material is concentrated and packaged into particles of smaller particle size. Positively charged cationic liposomes can concentrate and load gene drug siRNA into nanoparticles with smaller particle size. Cationic liposomes have shown incomparable advantages over other types of liposomes in gene delivery carrier preparation and cell transfection. The smaller particle size of the complex reduces the chance of being recognized, phagocytized, and cleared by macrophages in the body, and improves the bioavailability of the drug in vivo. At the same time, for tumor tissue, the smaller particle size of the complex is easier to penetrate from the gap of vascular endothelial cells into the tumor parenchyma through the penetration and retention effects, increasing the accumulation of drugs in tumor tissue. In terms of transfection, since the cell surface is slightly negatively charged, positively charged liposomes are more likely to adsorb to the cell surface and enter cells through mechanisms such as endocytosis, greatly increasing the transfection capacity of liposomes.
目前阳离子脂质作为基因载体因其结构简单、操作简便、生物安全性高等特点成为了目前应用最为广泛的非病毒载体，但大部分制备过程复杂，不易进行放大生产。因此本发明尝试利用3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸，以其为结构基础，将其两亲性衍生物作为基因药物的载体系统。此类衍生物合成步骤简单，原材料简单易得，同时，能较好递送基因药物，成功解决了上述问题，达到了较好的基因治疗效果。At present, cationic lipids as gene carriers have become the most widely used non-viral vectors due to their simple structure, easy operation, and high biological safety. However, most of the preparation processes are complicated and difficult to scale up. Therefore, the present invention attempts to use 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid as a structural basis and use its amphiphilic derivatives as a carrier system for gene medicines. The synthetic steps of such derivatives are simple, and the raw materials are simple and easy to obtain. At the same time, they can better deliver gene drugs, successfully solve the above problems, and achieve better gene therapy effects.
发明内容Contents of the invention
本发明的目的在于克服上述现有技术存在的缺点，提供一种3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸的两亲性衍生物及其用途。该衍生物是基于3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸结构为基础的一系列两亲性衍生化合物。本发明的两亲性衍生化合物的含氮头基在不同pH下具有不同的电离状态，其中在较低pH下，如pH4-5，其带正电。从而其制备而成的胶束及脂质体，可以与带负电的基因药物复合，能形成粒径较小，分布均匀的复合物纳米粒子。同时，化合物的含氮头基在pH7.4条件下不带电或带负电，这样使得复合物在pH7.4环境下不带电或带负电，降低了与体内血液环境中带负电蛋白吸附的机会，增加了脂质复合物的体内稳定性，减小因过多正电荷引起的细胞毒性。本发明提供的脂质体，可将荧光素酶siRNA体外高效递送到人非小细胞肺癌H1299-Pgl3细胞内，特异性抑制基因表达。同时载体系统可以体内特异性转载荧光基因药物进入正常小鼠肝脏细胞。The object of the present invention is to overcome the shortcomings of the above-mentioned prior art, and provide an amphiphilic derivative of 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid and its application. The derivative is a series of amphiphilic derivative compounds based on the structure of 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid. The nitrogen-containing headgroups of the amphiphilic derivative compounds of the present invention have different ionization states at different pH, wherein at lower pH, such as pH 4-5, they are positively charged. Therefore, the prepared micelles and liposomes can be compounded with negatively charged gene drugs to form composite nanoparticles with smaller particle size and uniform distribution. At the same time, the nitrogen-containing head group of the compound is uncharged or negatively charged at pH 7.4, which makes the complex uncharged or negatively charged at pH 7.4, reducing the chance of adsorption with negatively charged proteins in the blood environment in vivo. Increase the in vivo stability of lipoplexes and reduce the cytotoxicity caused by excessive positive charges. The liposome provided by the invention can efficiently deliver luciferase siRNA into human non-small cell lung cancer H1299-Pgl3 cells in vitro, and specifically inhibit gene expression. At the same time, the carrier system can specifically transfer fluorescent gene drugs into normal mouse liver cells in vivo.
本发明的目的是通过以下技术方案来实现的：The purpose of the present invention is achieved through the following technical solutions:
第一方面，本发明涉及一种3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸的两亲性衍生物，其结构式如式(Ⅰ)所示：In a first aspect, the present invention relates to an amphiphilic derivative of 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid, the structural formula of which is shown in formula (I):
(Ⅰ)，其中，n＝1、2、3或4，R1为长碳链烷基或烷烃基，R2为长碳链烷基或烷烃基。也可以表示为：R1为碳链或亚油酸链，R2为碳链或亚油酸链。其结构如具有以下特点：二甲氨基乙烷基-(甲基)氨基-(CH2)n-酯键-R1and R2。 (I), wherein, n=1, 2, 3 or 4, R1 is a long carbon chain alkyl or alkane group, and R2 is a long carbon chain alkyl or alkane group. It can also be expressed as: R1 is a carbon chain or a linoleic acid chain, and R2 is a carbon chain or a linoleic acid chain. Its structure has the following characteristics: dimethylaminoethyl-(methyl)amino-(CH2)n-ester bond-R1and R2.
优选地，所述R1为含12～18个碳原子的长碳链烷基或烷烃基，R2为含12～18个碳原子的长碳链烷基或烷烃基。Preferably, the R1 is a long carbon chain alkyl or alkane group containing 12 to 18 carbon atoms, and R2 is a long carbon chain alkyl or alkane group containing 12 to 18 carbon atoms.
更优选地，所述R1为第九个碳原子位置上为双键或第九、十二个碳原子位置上为双键的长碳链烷烃基。More preferably, the R1 is a long carbon chain alkane group with a double bond at the ninth carbon atom or a double bond at the ninth and twelfth carbon atoms.
更优选地，所述R2为第九个碳原子位置上为双键或第九、十二个碳原子位置上为双键的长碳链烷烃基。More preferably, the R2 is a long carbon chain alkane group with a double bond at the ninth carbon atom or a double bond at the ninth and twelfth carbon atoms.
上述两亲性衍生物包括含如下结构的各物质：The above-mentioned amphiphilic derivatives include substances with the following structures:
优选地，所述R1、R2为相同的基团。Preferably, the R1 and R2 are the same group.
优选地，其结构式如式(Ⅱ)所示：Preferably, its structural formula is shown in formula (II):
第二方面，本发明涉及一种上述的两亲性衍生物的制备方法，所述方法包括如下步骤：In a second aspect, the present invention relates to a method for preparing the aforementioned amphiphilic derivative, the method comprising the following steps:
A、在四氢呋喃催化剂、红铝溶液的甲苯溶液存在的条件下，含碳原子16-18的直链脂肪酸在室温下发生还原反应，得中间产物醇2；A, under the condition that the toluene solution of tetrahydrofuran catalyst, red aluminum solution exists, the straight-chain fatty acid containing carbon atom 16-18 takes place reduction reaction at room temperature, obtains intermediate product alcohol 2;
B、在三乙胺、4-二甲氨基吡啶、甲烷磺酸酐催化剂的作用下，中间产物醇在20℃下发生磺酸酯化反应得到中间产物3；B. Under the action of triethylamine, 4-dimethylaminopyridine and methanesulfonic anhydride catalyst, the intermediate product alcohol undergoes sulfonate reaction at 20°C to obtain intermediate product 3;
C、在二甲基甲酰胺、溴化锂催化剂作用下，中间产物3在45℃下反应得到中间溴化物4；C. Under the action of dimethylformamide and lithium bromide catalyst, the intermediate product 3 was reacted at 45° C. to obtain the intermediate bromide 4;
D、在镁、无水乙醚、甲酸乙酯催化剂作用下，中间产物4在40℃发生格式反应生成中间产物5；D. Under the catalyst action of magnesium, anhydrous ether and ethyl formate, the intermediate product 4 undergoes Grignard reaction at 40° C. to generate the intermediate product 5;
E、在氢氧化钠、四氢呋喃作用下，中间产物5在65℃下发生水解反应生成中间产物6；E. Under the action of sodium hydroxide and tetrahydrofuran, the intermediate product 5 undergoes a hydrolysis reaction at 65°C to generate the intermediate product 6;
F、在N，N-二异丙基乙胺、4-二甲氨基吡啶、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐催化剂作用下，中间产物6在50℃下发生酯化反应产生中间产物8；F, under the action of N, N-diisopropylethylamine, 4-dimethylaminopyridine, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride catalyst, the intermediate product 6 undergoes an esterification reaction at 50°C to produce an intermediate product 8;
G、在二甲基甲酰胺、碘化钠、碳酸钾的催化剂作用下，中间产物8在80℃下与N、N、N’-乙二胺发生氨基化反应，即得所述两亲性衍生物。G. Under the catalyst action of dimethylformamide, sodium iodide, and potassium carbonate, the intermediate product 8 undergoes amination reaction with N, N, N'-ethylenediamine at 80°C to obtain the amphiphilic derivative.
上述含碳原子12-18的直链脂肪酸结构式为：其中m＝1-7中任一整数，且第6个碳原子处为单键或双键，第9个碳原子处为单键或双键。The above-mentioned straight-chain fatty acid structural formula containing carbon atoms 12-18 is: Wherein, m=any integer from 1 to 7, and the 6th carbon atom is a single bond or double bond, and the 9th carbon atom is a single bond or a double bond.
第三方面，本发明涉及一种含上述的两亲性衍生物的脂质体，所述脂质体包括所述两亲性衍生物、胆碱、胆固醇；所述脂质体中胆碱的重量百分比含量为20％～50％，胆固醇的重量百分比含量为10％～30％。In a third aspect, the present invention relates to a liposome containing the above-mentioned amphiphilic derivative, wherein the liposome includes the amphiphilic derivative, choline, and cholesterol; the content of choline in the liposome The weight percentage content is 20%-50%, and the weight percentage content of the cholesterol is 10%-30%.
优选地，所述胆碱为二棕榈酰磷脂酰胆碱。Preferably, the choline is dipalmitoylphosphatidylcholine.
第四方面，本发明涉及一种上述的脂质体的制备方法，所述方法包括如下步骤：In a fourth aspect, the present invention relates to a method for preparing the above-mentioned liposome, said method comprising the steps of:
A、将所述两亲性衍生物的乙醇溶液、胆碱乙醇溶液、胆固醇乙醇溶液混合，形成脂质乙醇溶液；A. Mix the ethanol solution, choline ethanol solution, and cholesterol ethanol solution of the amphiphilic derivative to form a lipid ethanol solution;
B、将所述脂质乙醇溶液加入到pH<5.0的HEPES缓冲液中，搅拌，制得脂质体混悬液；B, the lipid ethanol solution is added into the HEPES buffer of pH<5.0, stirred, and the liposome suspension is obtained;
C、将所述脂质体混悬液在pH<5.0的HEPES缓冲液透析，去除乙醇；即得所述脂质体。C. Dialyzing the liposome suspension in HEPES buffer solution with pH<5.0 to remove ethanol; the liposome is obtained.
步骤B中，脂质体混悬液中含30wt.％乙醇。步骤C中，透析是在4℃下透析4h。In step B, the liposome suspension contains 30wt.% ethanol. In step C, the dialysis is performed at 4° C. for 4 hours.
第五方面，本发明涉及一种上述的脂质体在制备基因药物输送载体中的用途，所述脂质体包载DNA、RNA、透明质酸或多肽。所述基因药物为DNA、RNA、透明质酸或多肽。该脂质体具有帮助基因药物穿过细胞外障碍和细胞内障碍的能力。In the fifth aspect, the present invention relates to the use of the above-mentioned liposome in the preparation of a gene drug delivery carrier, the liposome encapsulating DNA, RNA, hyaluronic acid or polypeptide. The gene medicine is DNA, RNA, hyaluronic acid or polypeptide. The liposome has the ability to help gene medicines pass through extracellular barriers and intracellular barriers.
第六方面，本发明涉及一种脂质/基因复合物，所述脂质/基因复合物是由上述的脂质体包载基因生物分子而组成的。其包封率在30％以上。该基因生物分子包括DNA、RNA、透明质酸或多肽。In the sixth aspect, the present invention relates to a lipid/gene complex, which is composed of the above-mentioned liposomes encapsulating gene biomolecules. Its encapsulation rate is above 30%. The gene biomolecule includes DNA, RNA, hyaluronic acid or polypeptide.
第七方面，本发明涉及一种上述的脂质/基因复合物的制备方法，所述方法包括如下步骤：In a seventh aspect, the present invention relates to a method for preparing the above-mentioned lipid/gene complex, said method comprising the steps of:
A、将所述两亲性衍生物的乙醇溶液、胆碱乙醇溶液、胆固醇乙醇溶液混合，形成脂质乙醇溶液；A. Mix the ethanol solution, choline ethanol solution, and cholesterol ethanol solution of the amphiphilic derivative to form a lipid ethanol solution;
B、将所述脂质乙醇溶液加入到pH<5.0的HEPES缓冲液中，搅拌，制得脂质体混悬液；B, the lipid ethanol solution is added into the HEPES buffer of pH<5.0, stirred, and the liposome suspension is obtained;
C、在所述脂质体混悬液中加入基因生物分子的DEPC水溶液，37℃孵育1-2h；C. Add DEPC aqueous solution of gene biomolecules to the liposome suspension, and incubate at 37°C for 1-2h;
D、在pH<5.0的HEPES缓冲液透析，去除乙醇；继续在PBS溶液透析，调整体系pH至中性，即得所述脂质/基因复合物。D. Dialyze in HEPES buffer solution with pH<5.0 to remove ethanol; continue to dialyze in PBS solution to adjust the pH of the system to neutral to obtain the lipid/gene complex.
步骤B中，脂质体混悬液中含30wt.％乙醇。步骤D中，在HEPES缓冲液中透析是在4℃下透析4h；在PBS溶液中透析是在4℃下透析12h；其中PBS溶液的pH值为7.4。步骤D中，优选调整体系pH至7.4。In step B, the liposome suspension contains 30wt.% ethanol. In step D, the dialysis in the HEPES buffer was performed at 4° C. for 4 hours; the dialysis in the PBS solution was performed at 4° C. for 12 hours; wherein the pH of the PBS solution was 7.4. In step D, the pH of the system is preferably adjusted to 7.4.
与现有技术相比，本发明的有益效果如下：Compared with the prior art, the beneficial effects of the present invention are as follows:
(1)本发明基于3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸结构为基础，制备的两亲性衍生化合物，从而改善优化了传统基因递送系统合成步骤复杂，不易放大生产的问题；(1) The present invention is based on the structure of 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid, and prepares amphiphilic derivative compounds, thereby improving and optimizing the synthesis of traditional gene delivery systems The steps are complicated, and it is not easy to enlarge the production problem;
(2)本发明的两亲性衍生物制备而成的脂质体，在酸性pH下能带较多正电荷，增加了对基因药物siRNA的装载能力，复合后能形成粒径较小，分布均匀的复合物。同时在pH7.4环境下不带电或带负电，增加了脂质复合物的体内稳定性，减小因过多正电荷引起的细胞毒性，其可以作为基因药物载体对siRNA实现安全、有效递送；(2) The liposomes prepared from the amphiphilic derivatives of the present invention can carry more positive charges at acidic pH, which increases the loading capacity of gene drug siRNA, and can form a smaller particle size after compounding. homogeneous compound. At the same time, it is uncharged or negatively charged in a pH7.4 environment, which increases the stability of the lipoplex in vivo and reduces the cytotoxicity caused by excessive positive charges. It can be used as a gene drug carrier to achieve safe and effective delivery of siRNA;
(3)本发明的两亲性衍生物制备而成的脂质体，可体外有效转载基因药物进入细胞，特异性沉默目的基因；(3) Liposomes prepared from the amphiphilic derivatives of the present invention can effectively transfer gene drugs into cells in vitro, and specifically silence target genes;
(4)本发明的两亲性衍生物制备而成的脂质体，在溶酶体pH4.0的酸性条件下，化合物电离带正电，从而可以与溶酶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。同时，可体内有效转载基因药物经过血液循环及细胞内障碍，进入肝脏细胞。(4) The liposome prepared from the amphiphilic derivative of the present invention, under the acidic condition of lysosome pH 4.0, the compound is ionized and charged positively, so that it can interact with the phospholipid anion in the lysosome membrane, Form ion pairs that adapt to the non-bilayer structure, and then destroy the endosomal membrane to achieve endosomal escape. At the same time, it can effectively transfer gene drugs in vivo to enter liver cells through blood circulation and intracellular barriers.
附图说明Description of drawings
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:
图1为3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物TMEA的结构图；Fig. 1 is the structural diagram of 3-((2-(dimethylamino) ethyl group) (methyl) amino) propionic acid amphiphilic derivative TMEA;
图2为3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物TMEA的反应流程示意图；Fig. 2 is the schematic diagram of the reaction process of 3-((2-(dimethylamino) ethyl) (methyl) amino) propionic acid amphiphilic derivative TMEA;
图3为3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物TMEA的质谱图；Fig. 3 is the mass spectrogram of 3-((2-(dimethylamino) ethyl) (methyl) amino) propionic acid amphiphilic derivative TMEA;
图4为3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物TMEA的核磁图；Fig. 4 is the nuclear magnetic pattern of 3-((2-(dimethylamino) ethyl group) (methyl) amino) propionic acid amphiphilic derivative TMEA;
图5为3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物T1的结构图；Figure 5 is a structural diagram of 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid amphiphilic derivative T1;
图6为3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物T2的结构图；Figure 6 is a structural diagram of 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid amphiphilic derivative T2;
图7为TMEA脂质/luciferase-siRNA复合物凝胶阻滞电泳考察包封率示意图；Figure 7 is a schematic diagram of the encapsulation efficiency of TMEA lipid/luciferase-siRNA complex gel retardation electrophoresis;
图8为T1脂质/luciferase-siRNA复合物凝胶阻滞电泳考察包封率示意图；Figure 8 is a schematic diagram of the encapsulation efficiency of T1 lipid/luciferase-siRNA complex gel retardation electrophoresis;
图9为T2脂质/luciferase-siRNA复合物凝胶阻滞电泳考察包封率示意图；Figure 9 is a schematic diagram of the encapsulation efficiency of T2 lipid/luciferase-siRNA complex gel retardation electrophoresis;
图10为TMEA脂质/luciferase-siRNA复合物H1299细胞转染基因沉默结果示意图；Figure 10 is a schematic diagram of the gene silencing results of TMEA lipid/luciferase-siRNA complex transfection in H1299 cells;
图11为TMEA脂质/luciferase-siRNA复合物H1299细胞转染BCA蛋白结果示意图；Figure 11 is a schematic diagram of the results of BCA protein transfection of TMEA lipid/luciferase-siRNA complex H1299 cells;
图12为T1脂质/luciferase-siRNA复合物H1299细胞转染基因沉默示意图；Figure 12 is a schematic diagram of T1 lipid/luciferase-siRNA complex H1299 cell transfection gene silencing;
图13为T1脂质/luciferase-siRNA复合物H1299细胞转染BCA蛋白结果示意图；Figure 13 is a schematic diagram of the results of BCA protein transfection of T1 lipid/luciferase-siRNA complex H1299 cells;
图14为T2脂质/luciferase-siRNA复合物H1299细胞转染基因沉默示意图；Figure 14 is a schematic diagram of T2 lipid/luciferase-siRNA complex H1299 cell transfection gene silencing;
图15为T2脂质/luciferase-siRNA复合物H1299细胞转染BCA蛋白结果示意图；Figure 15 is a schematic diagram of the results of BCA protein transfection in T2 lipid/luciferase-siRNA complex H1299 cells;
图16为TMEA脂质/Cy-5-siRNA复合物在体内给药后的肝脏细胞分布示意图；Figure 16 is a schematic diagram of the liver cell distribution of the TMEA lipid/Cy-5-siRNA complex after administration in vivo;
图17为T1脂质/Cy-5-siRNA复合物在体内给药后的肝脏细胞分布示意图；Figure 17 is a schematic diagram of the distribution of liver cells after the T1 lipid/Cy-5-siRNA complex is administered in vivo;
图18为T2脂质/Cy-5-siRNA复合物在体内给药后的肝脏细胞分布示意图。Figure 18 is a schematic diagram of the distribution of T2 lipid/Cy-5-siRNA complexes in liver cells after administration in vivo.
具体实施方式detailed description
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按章制造厂商所建议的条件。The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention. For the test methods that do not specify specific conditions in the following examples, usually follow the conventional conditions, or the conditions suggested by the manufacturer.
实施例1、3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物TMEAExample 1, 3-((2-(dimethylamino) ethyl) (methyl) amino) propionic acid amphiphilic derivative TMEA
图1为两亲性化合物TMEA结构图；其制备如图2所示，具体为：选用N、N、N’-乙二胺、亚油酸等作为原料，按照反应流程图2合成，得到TMEA脂质。图3为两亲性化合物TMAEA进行核磁共振图谱结果，图4为其质谱图谱结果。Figure 1 is a structural diagram of the amphiphilic compound TMEA; its preparation is shown in Figure 2, specifically: select N, N, N'-ethylenediamine, linoleic acid, etc. as raw materials, synthesize according to the reaction scheme 2, and obtain TMEA Lipid. Fig. 3 is the result of the NMR spectrum of the amphiphilic compound TMAEA, and Fig. 4 is the result of the mass spectrogram.
反应原料：Reaction materials:
亚油酸、四氢呋喃(THF)、红铝溶液(vitride)、甲苯(toluene)、无水硫酸钠、乙酸乙酯、纯水、二氯甲烷、三乙胺、DMAP(4-二甲氨基吡啶)、EDC·HCl(1-(3-二甲基氨基丙基)-3-乙基碳化二亚胺盐酸盐)、甲烷磺酸酐、PMA(丙二醇甲醚醋酸酯)、盐酸、硫酸、氯化钠、DMF(二甲基甲酰胺)、溴化锂(LiBr)、正己烷、无水乙醚、二溴甲烷、Mg、甲酸乙酯、丙酮、NaOH、硅胶、DMAP、EDC·HCl、DCM(二氯甲烷)、DIPEA(N，N-二异丙基乙胺)、溴丙酸、Trans-4-(氨甲基)环己烷甲酸、甲醛、盐酸、(N、N、N’)-三甲基乙二胺、碳酸钾、DMF、NaILinoleic acid, tetrahydrofuran (THF), red aluminum solution (vitride), toluene (toluene), anhydrous sodium sulfate, ethyl acetate, pure water, dichloromethane, triethylamine, DMAP (4-dimethylaminopyridine) , EDC·HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), methanesulfonic anhydride, PMA (propylene glycol methyl ether acetate), hydrochloric acid, sulfuric acid, chlorination Sodium, DMF (dimethylformamide), lithium bromide (LiBr), n-hexane, anhydrous ether, dibromomethane, Mg, ethyl formate, acetone, NaOH, silica gel, DMAP, EDC·HCl, DCM (dichloromethane) , DIPEA (N,N-diisopropylethylamine), bromopropionic acid, Trans-4-(aminomethyl)cyclohexanecarboxylic acid, formaldehyde, hydrochloric acid, (N,N,N')-trimethylethane Diamine, potassium carbonate, DMF, NaI
步骤一：step one:
将30g亚油酸(反应物1)及THF(320ml)加入反应器中。将73ml的红铝溶液(60％wt/vol)的甲苯溶液缓慢滴加至反应器中，滴加过程中保持温度在0℃左右。滴加完毕后，室温反应2小时。溶液冷却至0℃，缓慢加入饱和硫酸钠溶液。滴加完毕后，30min内滴入130ml乙酸乙酯，并剧烈搅拌。反应液过滤，固体用乙酸乙酯冲洗，合并有机相，浓缩。产物溶解于80ml乙酸乙酯中，用水洗两次，并用无水硫酸钠干燥。过滤，浓缩有机相除去溶剂，得到产品2(28.8g)。30 g of linoleic acid (reactant 1) and THF (320 ml) were added to the reactor. 73ml of red aluminum solution (60%wt/vol) in toluene was slowly added dropwise to the reactor, and the temperature was kept at about 0°C during the dropwise addition. After the dropwise addition was completed, the reaction was carried out at room temperature for 2 hours. The solution was cooled to 0°C, and saturated sodium sulfate solution was slowly added. After the dropwise addition was completed, 130ml of ethyl acetate was added dropwise within 30 minutes and vigorously stirred. The reaction solution was filtered, the solid was rinsed with ethyl acetate, and the organic phases were combined and concentrated. The product was dissolved in 80 ml of ethyl acetate, washed twice with water, and dried over anhydrous sodium sulfate. Filtration and concentration of the organic phase to remove solvent gave product 2 (28.8 g).
步骤二：Step two:
500ml反应器中加入25g产物2及210ml二氯甲烷(DCM)，然后加入53ml三乙胺及1.15g DMAP(2.0mol)，溶液冷却至-10℃。32.7g甲烷磺酸酐溶于45ml DCM中，缓慢滴加至反应器中，并保持反应液温度在0℃以下。滴加完毕，继续保持0℃反应1小时。反应完毕后，80ml冰水加入反应液中，水相用DCM抽提。合并有机相，有机相用稀盐酸，水及饱和盐水洗涤，无水硫酸钠干燥。过滤，浓缩有机相除去有机溶剂，得到产品3(32.3g)。25g of product 2 and 210ml of dichloromethane (DCM) were added to a 500ml reactor, followed by 53ml of triethylamine and 1.15g of DMAP (2.0mol), and the solution was cooled to -10°C. 32.7g of methanesulfonic anhydride was dissolved in 45ml of DCM, slowly added dropwise to the reactor, and the temperature of the reaction solution was kept below 0°C. After the dropwise addition was completed, the reaction was continued at 0°C for 1 hour. After the reaction was completed, 80ml of ice water was added to the reaction solution, and the aqueous phase was extracted with DCM. The organic phases were combined, washed with dilute hydrochloric acid, water and saturated brine, and dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated to remove the organic solvent to obtain product 3 (32.3 g).
步骤三：Step three:
玻璃反应器中110ml DMF及30g产品3，冷却至-10℃。11.5g LiBr溶于110ml DMF，搅拌并缓慢滴加至反应器中，并保持反应液温度在0℃以下。滴加完毕后，反应液升温至45℃，搅拌过夜。反应完毕后，加入300ml水，并用240ml正己烷抽提，水相继续用2*45ml正己烷抽提。合并有机相，用水及饱和盐水洗涤，硫酸钠(17g)干燥。过滤，浓缩有机相除去有机溶剂，得到粗品27.5g。用60-120目硅胶纯化(正己烷为流动相)，得到纯品4约23g。110ml of DMF and 30g of product 3 in a glass reactor, cooled to -10°C. Dissolve 11.5g LiBr in 110ml DMF, stir and slowly drop into the reactor, and keep the temperature of the reaction solution below 0°C. After the dropwise addition, the temperature of the reaction solution was raised to 45° C. and stirred overnight. After the reaction was completed, 300ml of water was added and extracted with 240ml of n-hexane, and the aqueous phase was further extracted with 2*45ml of n-hexane. The organic phases were combined, washed with water and saturated brine, and dried over sodium sulfate (17 g). After filtration, the organic phase was concentrated to remove the organic solvent to obtain 27.5 g of crude product. Purify with 60-120 mesh silica gel (n-hexane as mobile phase) to obtain about 23 g of pure product 4.
步骤四：Step four:
三口烧瓶中加入2.21gMg及12ml无水乙醚。反应器中充入氩气。20g产物4溶解于40ml无水乙醚中。氩气保护下，8ml该溶液滴加至反应器中，并继续加入0.2ml二溴甲烷。反应液在水浴中升温至40℃。反应开始后，移去热源，将剩余32ml溶液滴加至反应器中，让混合物保持轻微回流状态。滴加完毕后，加热使其保持回流状态反应。反应完毕后，反应液用冰浴冷却至10℃以下，然后缓慢加入甲酸乙酯的乙醚溶液(2.2ml溶于32ml乙醚中)。滴加完毕后，室温反应过夜。然后加入56ml冰水及10％的硫酸溶液，分离有机相，水相用乙醚抽提。合并有机相，用盐水洗涤，硫酸钠干燥。过滤，浓缩有机相除去有机溶剂，得到粗品(醇及甲酸酯混合物)16g。粗品用100mlTHF溶解，加入NaOH溶液(7.5g溶于150ml水中)，加热至65℃反应18小时。反应完全后，反应物冷却至室温，并用乙醚抽提，合并有机相，用40ml盐水洗涤。硫酸钠干燥。过滤，浓缩有机相。粗品用60-120目硅胶纯化(4％乙醚/正己烷)，得到纯品DLM6a(11.6g)(产率40％)。Add 2.21g of Mg and 12ml of anhydrous diethyl ether into the three-necked flask. The reactor was filled with argon. 20 g of product 4 was dissolved in 40 ml of anhydrous ether. Under the protection of argon, 8 ml of this solution was added dropwise into the reactor, and 0.2 ml of dibromomethane was added continuously. The temperature of the reaction solution was raised to 40° C. in a water bath. After the reaction started, the heat source was removed, and the remaining 32ml of the solution was added dropwise into the reactor, and the mixture was kept in a slight reflux state. After the dropwise addition is complete, heat to keep it under reflux to react. After the reaction was completed, the reaction solution was cooled to below 10°C with an ice bath, and then a solution of ethyl formate in ether (2.2ml dissolved in 32ml ether) was slowly added. After the dropwise addition was completed, the reaction was carried out overnight at room temperature. Then 56ml of ice water and 10% sulfuric acid solution were added, the organic phase was separated, and the aqueous phase was extracted with ether. The combined organic phases were washed with brine and dried over sodium sulfate. After filtration, the organic phase was concentrated to remove the organic solvent to obtain 16 g of crude product (alcohol and formate mixture). The crude product was dissolved in 100ml THF, NaOH solution (7.5g dissolved in 150ml water) was added, and heated to 65°C for 18 hours. After the reaction was complete, the reactant was cooled to room temperature and extracted with ether, and the organic phases were combined and washed with 40 ml of brine. Na2SO4 dried. Filter and concentrate the organic phase. The crude product was purified with 60-120 mesh silica gel (4% ether/n-hexane) to obtain pure DLM6a (11.6 g) (yield 40%).
步骤五：Step five:
三口烧瓶加入DLM1.1g和10ml DCM。依次加入溴丙酸477mg，EDC.HCL747mg，DMAP38mg，DIPEA0.8ml。反应器在油浴中升温至50℃，搅拌反应18-20小时。反应完毕后，15ml水加入反应液中，溶液分层，水相用DCM抽提。合并有机相，有机相用盐水洗涤，硫酸钠干燥。过滤，浓缩有机相除去有机溶剂，得到粗产品1.5g。用60-120目硅胶纯化(正己烷为流动相)，得到纯品8约800mg(57.8％)。Add DLM1.1g and 10ml DCM to the three-necked flask. Add bromopropionic acid 477mg, EDC.HCL747mg, DMAP38mg, DIPEA0.8ml successively. The temperature of the reactor was raised to 50°C in an oil bath, and the reaction was stirred for 18-20 hours. After the reaction was completed, 15ml of water was added to the reaction solution, the solution was separated into layers, and the aqueous phase was extracted with DCM. The combined organic phases were washed with brine and dried over sodium sulfate. After filtration, the organic phase was concentrated to remove the organic solvent to obtain 1.5 g of crude product. Purify with 60-120 mesh silica gel (n-hexane as mobile phase) to obtain about 800 mg (57.8%) of pure product 8.
步骤六：Step six:
三口烧瓶加入8(500)mg，加入DMF30ml使其完全溶解，依次加入N、N、N’-乙二胺(420mg)，NaI112mg，K2CO3311mg，反应器在油浴中升温至80℃，搅拌反应36小时。反应完毕后，加入水20ml，二氯甲烷萃取，合并有机相，，无水硫酸钠干燥、过滤。浓缩有机相除去有机溶剂，得到粗产品800mg。用60-120目硅胶纯化(1％甲醇/二氯甲烷为流动相)，得到纯品13(160mg)(31％)。Add 8 (500) mg to the three-necked flask, add 30 ml of DMF to dissolve it completely, add N, N, N'-ethylenediamine (420 mg), NaI 112 mg, K2 CO3 311 mg in sequence, and the reactor is heated to 80°C in an oil bath , stirred for 36 hours. After the reaction was completed, 20 ml of water was added, extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated to remove the organic solvent to obtain 800 mg of crude product. Purification with 60-120 mesh silica gel (1% methanol/dichloromethane as mobile phase) afforded pure product 13 (160 mg) (31%).
对两亲性化合物MPZ进行质谱图谱、核磁图谱分析，由图3、4可知，所得MPZ化合物纯度大于95％，其分子量与理论值相近，为分子量685g/mol。The amphiphilic compound MPZ is subjected to mass spectrometry and nuclear magnetic spectrum analysis. It can be seen from Figures 3 and 4 that the purity of the obtained MPZ compound is greater than 95%, and its molecular weight is close to the theoretical value, which is a molecular weight of 685g/mol.
脂质化合物DLin-MC2-TMEA12：Lipid compound DLin-MC2-TMEA12:
MS(ES+):C45H84N2O2,calculated685.6533,found685.6627；MS (ES+ ): C45 H84 N2 O2 , calculated685.6533, found685.6627;
1HNMR(400MHz,CDCl3)δ5.36-5.39(8H)，4.9(1H)，2.72-2.79(4H)，2.48-2.51(9H)；2.29(4H)；2.04-2.07(8H)；1.51-1.52(8H)；1.28-1.37(36H)；0.89-0.92(6H)。1 HNMR (400MHz, CDCl3 ) δ5.36-5.39(8H), 4.9(1H), 2.72-2.79(4H), 2.48-2.51(9H); 2.29(4H); 2.04-2.07(8H); 1.51- 1.52 (8H); 1.28-1.37 (36H); 0.89-0.92 (6H).
实施例2、3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物T1Example 2, 3-((2-(dimethylamino) ethyl) (methyl) amino) propionic acid amphiphilic derivative T1
图5为3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物T1的结构图；其制备具体为：选用N、N、N’-乙二胺、棕榈酸等作为原料，按照实施例1合成方法合成，所不同之处在于：步骤1中，反应物1为棕榈酸，其他合成步骤、合成原理及原料完全相同。得到T1脂质；均经过HPLC纯化和质谱鉴定，纯度大于95％，分子量与理论值相符。Figure 5 is a structural diagram of 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid amphiphilic derivative T1; its preparation is as follows: select N, N, N'-ethyl Diamine, palmitic acid, etc. are used as raw materials and synthesized according to the synthesis method of Example 1, the difference is that in step 1, reactant 1 is palmitic acid, and other synthesis steps, synthesis principles and raw materials are exactly the same. T1 lipids were obtained; all were purified by HPLC and identified by mass spectrometry, the purity was greater than 95%, and the molecular weight was consistent with the theoretical value.
实施例3、3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物T2Example 3, 3-((2-(dimethylamino) ethyl) (methyl) amino) propionic acid amphiphilic derivative T2
图6为3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸两亲性衍生物T2结构图；其制备具体为：选用N、N、N’-乙二胺、油酸等作为原料，按照实施例1合成方法合成，所不同之处在于：步骤1中，反应物1为油酸，其他合成步骤、合成原理及原料完全相同。得到T2脂质。均经过HPLC纯化和质谱鉴定，纯度大于95％，分子量与理论值相符。Figure 6 is a structural diagram of 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid amphiphilic derivative T2; Amine, oleic acid, etc. are used as raw materials, and synthesized according to the synthesis method of Example 1, the difference is that in step 1, reactant 1 is oleic acid, and other synthesis steps, synthesis principles and raw materials are exactly the same. Obtain T2 lipid. All have been purified by HPLC and identified by mass spectrometry, the purity is greater than 95%, and the molecular weight is consistent with the theoretical value.
实施例4、T1脂质体Embodiment 4, T1 liposome
本实施例利用两亲性衍生化合物T1与辅助脂质，按照不同比例制备了脂质体。In this example, liposomes were prepared according to different ratios by using amphiphilic derivative compound T1 and auxiliary lipids.
其制备方法包括如下步骤：Its preparation method comprises the following steps:
1.制备样品溶液1. Preparation of Sample Solutions
T1乙醇溶液、二棕榈酰磷脂酰胆碱(DPPC)乙醇溶液、胆固醇(Cholesterol，CHOL)乙醇溶液配制：以电子天平称取一定量，加入无水乙醇使之成为10mg/ml,并以之作为贮备液；Preparation of T1 ethanol solution, dipalmitoylphosphatidylcholine (DPPC) ethanol solution, and cholesterol (Cholesterol, CHOL) ethanol solution: Weigh a certain amount with an electronic balance, add absolute ethanol to make it 10mg/ml, and use it as stock solution;
4-羟乙基哌嗪乙磺酸(HEPES)缓冲液(HEPES buffer)的配制：以电子天平称取HEPES，加入去离子水,用盐酸溶液调pH,使之成为5mM pH4.0，焦碳酸二乙酯(DEPC)处理，灭菌后，以之作为贮备液；Preparation of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer (HEPES buffer): Weigh HEPES with an electronic balance, add deionized water, adjust pH with hydrochloric acid solution to make it 5mM pH4.0, pyrocarbonic acid Diethyl ester (DEPC) treatment, after sterilization, use it as a stock solution;
2.脂质体的制备：2. Preparation of liposomes:
1)取出T1化合物,胆固醇(CHOL)，二棕榈酰磷脂酰胆碱(DPPC)储备液，室温下平衡半小时；1) Take out T1 compound, cholesterol (CHOL), dipalmitoylphosphatidylcholine (DPPC) stock solution, and equilibrate at room temperature for half an hour;
2)分别按一定摩尔比，量取T1乙醇溶液、DPPC乙醇溶液、CHOL乙醇溶液至5ml离心管混合；2) Measure T1 ethanol solution, DPPC ethanol solution, CHOL ethanol solution to a 5ml centrifuge tube according to a certain molar ratio;
3)取5Mm pH4.0HEPES Buffer于5ml离心管中，30℃预热，5-10min；将vortex涡旋仪调至shake II档，在涡旋搅拌下，将脂质乙醇混合液缓慢加入5mM pH4.0HEPES buffer中，涡旋搅拌1～2min，制备含30％乙醇的脂质体混悬液；3) Take 5Mm pH4.0HEPES Buffer in a 5ml centrifuge tube, preheat at 30°C for 5-10min; adjust the vortex to shake II, and slowly add the lipid-ethanol mixture to 5mM pH4 under vortex stirring .0 in HEPES buffer, vortex and stir for 1 to 2 minutes to prepare a liposome suspension containing 30% ethanol;
4)将步骤3)得到的脂质体混悬液首先置于5mM pH4.0HEPES buffer中4℃下透析12h出去乙醇。4) The liposome suspension obtained in step 3) was first placed in 5 mM pH4.0 HEPES buffer and dialyzed at 4°C for 12 hours to remove ethanol.
5)激光散射粒度分析仪(PCS)测定纳米粒子粒径分布、电动电势(zeta电位)，使用的仪器是英国马尔文公司的ZetaSizer3000H激光粒度仪，使用He-Ne离子激光(λ0＝633nm)为入射光，动力学光散射试验在25℃进行，反射角为1.33，角度为90°。连续检测三次的平均值作为得到的数据。5) laser scattering particle size analyzer (PCS) measures nanoparticle size distribution, zeta potential (zeta potential), and the instrument used is the ZetaSizer3000H laser particle size analyzer of Malvern, UK, using He-Ne ion laser (λ0=633nm) as For incident light, the dynamic light scattering test is carried out at 25°C, the reflection angle is 1.33, and the angle is 90°. The average value of three consecutive detections was taken as the obtained data.
效果验证：Effect verification:
通过对不同脂质组分组成、不同脂质组分比例制备的T1脂质体的表征如表1所示；The characterization of T1 liposomes prepared by different lipid component compositions and different lipid component ratios is shown in Table 1;
表1Table 1
实施例5、T2脂质体Embodiment 5, T2 liposome
本实施例利用两亲性衍生化合物T2与辅助脂质，按照不同比例制备了脂质体。In this example, liposomes were prepared according to different ratios by using amphiphilic derivative compound T2 and auxiliary lipids.
其制备及表征方法如实施例3脂质体制备方法。Its preparation and characterization methods are as in Example 3 liposome preparation method.
效果验证：Effect verification:
不同原料、不同比例制备的T2脂质体的表征如下表2所示：The characterization of T2 liposomes prepared with different raw materials and different ratios is shown in Table 2 below:
表2Table 2
实施例6、TMEA脂质体Embodiment 6, TMEA liposome
本实施例利用两亲性衍生化合物TMEA与辅助脂质，按照不同比例制备了脂质体。In this example, liposomes were prepared according to different ratios by using the amphiphilic derivative compound TMEA and auxiliary lipids.
其制备及表征方法如实施例3脂质体制备方法。Its preparation and characterization methods are as in Example 3 liposome preparation method.
效果验证：Effect verification:
不同原料、不同比例制备的TMEA脂质体的表征如下表3所示：The characterization of the TMEA liposomes prepared by different raw materials and different ratios is shown in Table 3 below:
表3table 3
实施例7、TMEA脂质体/基因复合物Embodiment 7, TMEA liposome/gene complex
本实施例利用TMEA脂质制备的脂质体，按照不同比例N/P与siRNA制备了脂质/基因复合物，并对其进行表征。In this example, liposomes prepared from TMEA lipids were used to prepare lipid/gene complexes according to different ratios of N/P and siRNA, and to characterize them.
其方法如下：The method is as follows:
1.空白脂质体制备：制备方法及处方比例如实施例3脂质体制备方法；1. Blank liposome preparation: preparation method and prescription ratio are as embodiment 3 liposome preparation method;
2.脂质/基因复合物制备：2. Lipid/gene complex preparation:
1)配制小干扰RNA(siRNA)溶液：将siRNA用DEPC水溶解，使之成为1mg/ml，并以之作为贮备液；1) Prepare a small interfering RNA (siRNA) solution: dissolve siRNA in DEPC water to make it 1 mg/ml, and use it as a stock solution;
2)按不同siRNA/脂质总量(N/P)比，取适量siRNA溶液，将未透析的脂质体混悬液放置在vortex涡旋仪上，缓慢加入siRNA溶液，37℃孵育1-2h；2) According to different siRNA/lipid total (N/P) ratios, take an appropriate amount of siRNA solution, place the undialyzed liposome suspension on a vortex, slowly add siRNA solution, and incubate at 37°C for 1- 2h;
3)将制备好的复合物置于5mM pH4.0HEPES buffer中，4℃下透析4h，除去体系中的乙醇。然后取出，继续在0.01M pH7.4PBS溶液中，4℃下透析12h，将体系pH调为7.4，接近体液pH，即为脂质基因复合物。透析结束收集于1.5ml离心管中，4℃保存备用；3) The prepared complex was placed in 5 mM pH4.0 HEPES buffer, dialyzed at 4° C. for 4 h, and ethanol in the system was removed. Then take it out, and continue to dialyze in 0.01M pH7.4 PBS solution at 4°C for 12 hours, and adjust the pH of the system to 7.4, which is close to the pH of body fluids, which is the lipid gene complex. After dialysis, collect in a 1.5ml centrifuge tube and store at 4°C for later use;
3.脂质/基因复合物在不同pH环境下的带电情况及粒子均一度以及对siRNA的包封情况表征：3.Characterization of lipid/gene complexes in different pH environments, particle uniformity and encapsulation of siRNA:
1)激光散射粒度分析仪(PCS)测定在0.01M pH7.4PBS中透析除去乙醇的脂质/基因复合物的粒径分布及粒子zeta电位，考察复合物在pH7.4中性条件下的带电情况及粒径分布；1) Laser scattering particle size analyzer (PCS) was used to measure the particle size distribution and particle zeta potential of the lipid/gene complex dialyzed to remove ethanol in 0.01M pH7.4PBS, and to investigate the charge of the complex at pH7.4 neutral condition condition and particle size distribution;
2)激光散射粒度分析仪(PCS)测定在5mM pH4.0HEPES buffer中透析除去乙醇的脂质/基因复合物的粒径分布及粒子zeta电位，考察复合物在pH4.0条件下的带电情况及粒径分布；2) Laser scattering particle size analyzer (PCS) was used to measure the particle size distribution and particle zeta potential of the lipid/gene complex dialyzed to remove ethanol in 5mM pH4.0 HEPES buffer, and to investigate the charged condition and Particle size distribution;
3)用凝胶电泳仪，利用利用“分子筛”和“电泳”的双重作用，分离未稳定包裹的siRNA，从条带结果分析对siRNA包封情况。3) Use a gel electrophoresis instrument to separate unstabilized siRNAs by using the dual functions of "molecular sieve" and "electrophoresis", and analyze the encapsulation of siRNAs from the results of the bands.
效果验证：Effect verification:
(1)不同原料比例、不同N/P比制备的TMEA脂质体/基因复合物的表征如表4所示，由结果可以看出，TMEA两亲性化合物制备的脂质体包裹基因药物siRNA后，形成了粒径较小且分布均匀的复合纳米粒子。同时，复合物在近血液环境pH7.4的条件下为负电性，大大减小了阳离子脂质过剩的正电性引起的毒性。而包裹了siRNA的脂质/基因复合物在近核内体pH4.0的条件下带正电，说明其进入细胞内的核内体后可以因电荷反应与负电性的膜融合，逃逸出核内体，免受酶降解失活。同时，由图7可以看出从凝胶电泳阻滞结果来看，在100：1、50:1、20:1三个复合比下制备的复合物电泳条带几乎没有显示，而20:1组的复合物，脂质体被破膜后，观察有明显条带，因此说明TMEA脂质体对siRNA进行了完全包封，能保护其不受外界环境的破坏。因此，从以上两点可以看出，TMEA两亲性化合物制备的脂质体具有帮助基因药物穿过细胞外障碍和细胞内障碍的能力。(1) The characterization of the TMEA liposome/gene complexes prepared by different raw material ratios and different N/P ratios is shown in Table 4. As can be seen from the results, the liposome-encapsulated gene drug siRNA prepared by TMEA amphiphilic compounds Finally, composite nanoparticles with smaller particle size and uniform distribution are formed. At the same time, the complex is negatively charged at pH 7.4 near the blood environment, which greatly reduces the toxicity caused by the excess positive charge of cationic lipids. However, the lipid/gene complex encapsulating siRNA is positively charged at pH 4.0 in the perinuclear endosome, indicating that after entering the endosome in the cell, it can fuse with the negatively charged membrane due to a charge reaction and escape from the nucleus. Endosomes, protected from enzymatic degradation and inactivation. At the same time, it can be seen from Figure 7 that from the gel electrophoresis retardation results, the complex electrophoresis bands prepared under the three compounding ratios of 100:1, 50:1, and 20:1 hardly show, while the 20:1 After the liposomes were ruptured, obvious bands were observed, which indicated that the TMEA liposomes completely encapsulated the siRNA and could protect it from the damage of the external environment. Therefore, it can be seen from the above two points that liposomes prepared by TMEA amphiphilic compounds have the ability to help gene medicines pass through extracellular barriers and intracellular barriers.
表4Table 4
实施例8、T1脂质体/基因复合物Embodiment 8, T1 liposome/gene complex
本实施例利用T1两亲性化合物制备的脂质体，按照不同比例N/P与siRNA制备了脂质/基因复合物，并对其进行表征。In this example, liposomes prepared by T1 amphiphilic compounds were used to prepare lipid/gene complexes according to different ratios of N/P and siRNA, and to characterize them.
其方法如下：The method is as follows:
实验方法如实施例7复合物表征。The experimental method was as in Example 7 for compound characterization.
效果验证：Effect verification:
(1)不同原料比例、不同N/P比制备的T1脂质体/基因复合物的表征如表5所示，由结果可以看出，T1两亲性化合物制备的脂质体包裹基因药物siRNA后，形成了粒径较小且分布均匀的复合纳米粒子。同时，复合物在近血液环境pH7.4的条件下为负电性，大大减小了阳离子脂质过剩的正电性引起的毒性。而包裹了siRNA的脂质/基因复合物在近核内体pH4.0的条件下带正电，说明其进入细胞内的核内体后可以因电荷反应与负电性的膜融合，逃逸出核内体，免受酶降解失活。由图8的凝胶电泳阻滞结果来看，在100：1、50:1、20:1三个复合比下制备的复合物电泳条带几乎没有显示，而20:1组的复合物，脂质体被破膜后，观察有明显条带，因此说明MPZ脂质体对siRNA进行了完全包封，能保护其不受外界环境的破坏。因此，从以上可以看出，T1脂质制备的脂质体具有帮助基因药物穿过细胞外障碍和细胞内障碍的能力。(1) The characterization of the T1 liposome/gene complex prepared by different raw material ratios and different N/P ratios is shown in Table 5. It can be seen from the results that the liposome-encapsulated gene drug siRNA prepared by the T1 amphiphilic compound Finally, composite nanoparticles with smaller particle size and uniform distribution were formed. At the same time, the complex is negatively charged at pH 7.4 near the blood environment, which greatly reduces the toxicity caused by the excess positive charge of cationic lipids. However, the lipid/gene complex encapsulating siRNA is positively charged at pH 4.0 in the perinuclear endosome, indicating that after entering the endosome in the cell, it can fuse with the negatively charged membrane due to a charge reaction and escape from the nucleus. Endosomes, protected from enzymatic degradation and inactivation. From the results of gel electrophoresis retardation in Figure 8, the electrophoretic bands of the complexes prepared under the three compounding ratios of 100:1, 50:1, and 20:1 are almost not displayed, while the complexes in the 20:1 group, After the liposome was ruptured, obvious bands were observed, which indicated that the MPZ liposome completely encapsulated the siRNA and could protect it from the damage of the external environment. Therefore, it can be seen from the above that liposomes prepared from T1 lipids have the ability to help gene drugs pass through extracellular and intracellular barriers.
表5table 5
实施例9、T2脂质体/基因复合物Embodiment 9, T2 liposome/gene complex
本实施例利用T2两亲性化合物制备的脂质体，按照不同比例N/P与siRNA制备了脂质/基因复合物，并对其进行表征。In this example, liposomes prepared by T2 amphiphilic compounds were used to prepare lipid/gene complexes according to different ratios of N/P and siRNA, and then characterized them.
其方法如下：The method is as follows:
实验方法如实施例7复合物表征。The experimental method was as in Example 7 for compound characterization.
效果验证：Effect verification:
(1)不同原料比例、不同N/P比制备的T2脂质体/基因复合物的表征如表6所示，由结果可以看出，T2两亲性化合物制备的脂质体包裹基因药物siRNA后，形成了粒径较小且分布均匀的复合纳米粒子。同时，复合物在近血液环境pH7.4的条件下为负电性，大大减小了阳离子脂质过剩的正电性引起的毒性。而包裹了siRNA的脂质/基因复合物在近核内体pH4.0的条件下带正电，说明其进入细胞内的核内体后可以因电荷反应与负电性的膜融合，逃逸出核内体，免受酶降解失活。由图9的凝胶电泳阻滞结果来看，在100：1、50:1、20:1三个复合比下制备的复合物电泳条带几乎没有显示，而20:1组的复合物，脂质体被破膜后，观察有明显条带，因此说明MPZ脂质体对siRNA进行了完全包封，能保护其不受外界环境的破坏。因此，从以上可以看出，T1脂质制备的脂质体具有帮助基因药物穿过细胞外障碍和细胞内障碍的能力。因此，从以上可以看出，T2两亲性化合物制备的脂质体具有帮助基因药物穿过细胞外障碍和细胞内障碍的能力。(1) The characterization of the T2 liposome/gene complex prepared by different raw material ratios and different N/P ratios is shown in Table 6. It can be seen from the results that the liposome-encapsulated gene drug siRNA prepared by the T2 amphiphilic compound Finally, composite nanoparticles with smaller particle size and uniform distribution were formed. At the same time, the complex is negatively charged at pH 7.4 near the blood environment, which greatly reduces the toxicity caused by the excess positive charge of cationic lipids. However, the lipid/gene complex encapsulating siRNA is positively charged at pH 4.0 in the perinuclear endosome, indicating that after entering the endosome in the cell, it can fuse with the negatively charged membrane due to a charge reaction and escape from the nucleus. Endosomes, protected from enzymatic degradation and inactivation. From the results of gel electrophoresis retardation in Figure 9, the electrophoretic bands of the complexes prepared under the three compounding ratios of 100:1, 50:1, and 20:1 are almost not displayed, while the complexes in the 20:1 group, After the liposome was ruptured, obvious bands were observed, which indicated that the MPZ liposome completely encapsulated the siRNA and could protect it from the damage of the external environment. Therefore, it can be seen from the above that liposomes prepared from T1 lipids have the ability to help gene drugs pass through extracellular and intracellular barriers. Therefore, it can be seen from the above that the liposome prepared by the T2 amphiphilic compound has the ability to help gene medicine pass through extracellular barriers and intracellular barriers.
表6Table 6
实施例10、TMEA可电离阳离子脂质体/基因复合物人肺癌H1299-pGL3细胞转染效Embodiment 10, TMEA ionizable cationic liposome/gene complex human lung cancer H1299-pGL3 cell transfection efficiency果fruit
本实施例利用TMEA两亲性化合物制备的脂质体/luciferase-siRNA复合物对人非小细胞肺癌H1299细胞进行细胞转染，由其基因沉默效果，来观察载体脂质对基因药物的转运情况。In this example, the liposome/luciferase-siRNA complex prepared by TMEA amphiphilic compound was used to transfect human non-small cell lung cancer H1299 cells, and the gene silencing effect was used to observe the transfer of carrier lipids to gene drugs .
1.脂质/基因复合物的制备：1. Preparation of lipid/gene complexes:
TMEA/基因复合物制备方法如实施例7，以TMEA/DPPC/Chol-(40/40/30mol)摩尔比制备脂质体，按照N/P-20/1包裹荧光素酶luciferase-siRNA，制备脂质/基因复合物；The preparation method of TMEA/gene complex is as in Example 7, liposomes are prepared with TMEA/DPPC/Chol-(40/40/30mol) molar ratio, and luciferase luciferase-siRNA is wrapped according to N/P-20/1 to prepare lipid/gene complexes;
2.H1299细胞收集与培养：2. Collection and culture of H1299 cells:
人非小细胞肺癌H1299-pGL3细胞系由实验室传代获得，采用含10％小牛血清的RPMI1640培养基于37℃、5％CO2培养箱培养，2～4天更换培养基一次，1:3常规传代培养，取对数期细胞进行实验；The human non-small cell lung cancer H1299-pGL3 cell line was obtained by subculture in the laboratory. It was cultured in RPMI1640 containing 10% calf serum. It was cultured in a 37°C, 5% CO2 incubator. The medium was replaced every 2 to 4 days, and the routine was 1:3. For subculture, take logarithmic phase cells for experiments;
3.复合物与细胞孵育：3. Complex and cell incubation:
细胞在转染实验前24小时按105/Well接种于24孔板，18h后观察，约长到70-80％。转染前用不含血清的培养基洗细胞一次，将样品用适量Opti-MEM培养基稀释，以每孔400μl/well加入24孔板。置培养箱中转染2.5～4小时后，换成含血清培养基继续培养36-48小时后，荧光素酶检测仪(Luminometer)检测(RLU)值，测量报告基因的表达，BCA蛋白含量测定计算细胞板每孔蛋白含量，同时用空白PBS组，裸siRNA组作为对照，考察载体转染基因药物的能力；The cells were seeded in 24-well plates at 105 /Well 24 hours before the transfection experiment, and observed 18 hours later, the growth was about 70-80%. Wash the cells once with serum-free medium before transfection, dilute the sample with an appropriate amount of Opti-MEM medium, and add 400 μl/well per well to a 24-well plate. Place in the incubator for 2.5-4 hours after transfection, replace with serum-containing medium and continue to culture for 36-48 hours, then detect the (RLU) value with a luciferase detector (Luminometer), measure the expression of the reporter gene, and determine the BCA protein content Calculate the protein content of each well of the cell plate, and use the blank PBS group and the naked siRNA group as controls at the same time to investigate the ability of the vector to transfect the gene drug;
1)荧光素酶活力检测：1) Detection of luciferase activity:
Luciferase报告基因的转染结果的检测步骤按照Luciferase Assay System的操作说明测定，使用荧光素酶检测仪(Luminometer)检测相对发光值；The detection steps of the transfection results of the Luciferase reporter gene were determined according to the operation instructions of the Luciferase Assay System, and the relative luminescence value was detected by using a luciferase detector (Luminometer);
ⅰ.将5X CCLR(细胞裂解液)稀释成1X，取分装的荧光素酶底物(Luciferase AssaySubstance)待其恢复室温使用；ⅰ. Dilute 5X CCLR (cell lysate) to 1X, take the aliquoted luciferase substrate (Luciferase Assay Substance) and wait for it to return to room temperature for use;
ⅱ.将转染后的细胞去除培养基后用PBS润洗3次，润洗后将孔中的PBS吸干，然后每孔添加200μl的1X细胞裂解液于37℃30min；ⅱ. Remove the culture medium from the transfected cells and rinse with PBS for 3 times. After rinsing, blot the PBS in the wells to dry up, then add 200 μl of 1X cell lysate to each well for 30 minutes at 37°C;
ⅲ.充分裂解后将混合物移入离心管中，用13000rpm的转速离心3min，用上清液作为检测样品；ⅲ. After fully lysing, move the mixture into a centrifuge tube, centrifuge at a speed of 13000rpm for 3min, and use the supernatant as a test sample;
ⅳ.每个检测样品取10μl与10μl的荧光素酶底物在检测管中充分混合(用移液枪吹打10次)后放入荧光素酶检测仪(Luminometer)中测定Luciferase的发光值(RLU)。ⅳ. Take 10 μl and 10 μl of luciferase substrate for each test sample and mix them thoroughly in the test tube (by pipetting 10 times) and put them into a luciferase detector (Luminometer) to measure the luminescence value of Luciferase (RLU ).
2)BCA蛋白含量测定法：2) BCA protein content assay method:
每孔细胞蛋白浓度的检测步骤按照BCA Protein Assay Kit的操作说明测定，使用酶标仪检测光密度(OD)值；The detection steps of the cell protein concentration in each well were determined according to the operating instructions of the BCA Protein Assay Kit, and the optical density (OD) value was detected by a microplate reader;
ⅰ.绘制标准曲线；ⅰ. Draw a standard curve;
ⅱ.配置BCA工作试剂(WR)，在96孔板中每孔加入200μl的WR，然后像孔中加入10μl样品或者上述离心后的上清检测样品；ⅱ. Configure BCA working reagent (WR), add 200 μl of WR to each well of the 96-well plate, and then add 10 μl of sample or the above-mentioned centrifuged supernatant test sample to the well;
ⅲ.加入样品后，将96孔板至于37℃恒温恒湿箱中放置30min使其充分反应，再用酶标仪在562nm处测量光密度(OD)值；ⅲ. After adding the sample, place the 96-well plate in a constant temperature and humidity box at 37°C for 30 minutes to allow it to fully react, then measure the optical density (OD) value at 562nm with a microplate reader;
ⅳ.根据标准样品的光密度值绘制标准曲线，然后根据标准曲线拟合得到回归方程计算检测样品中的蛋白质含量。ⅳ. Draw a standard curve according to the optical density value of the standard sample, and then calculate the protein content in the test sample according to the regression equation obtained by fitting the standard curve.
效果验证：Effect verification:
(1)TMEA/luciferase-siRNA复合物粒径分布及均一度结果如表7所示；(1) The particle size distribution and uniformity of TMEA/luciferase-siRNA complexes are shown in Table 7;
(2)TMEA/luciferase-siRNA复合物转染H1299-pGL3细胞，基因沉默结果如图10所示；(2) TMEA/luciferase-siRNA complex was transfected into H1299-pGL3 cells, and the gene silencing results are shown in Figure 10;
(3)TMEA/luciferase-siRNA复合物转染H1299-pGL3细胞，BCA蛋白结果如图11所示；(3) TMEA/luciferase-siRNA complex was transfected into H1299-pGL3 cells, and the results of BCA protein were shown in Figure 11;
由结果中可以看出，经TMEA两亲性化合物制备的脂质体包裹siRNA进入了细胞，并特异性沉默了目的基因，与PBS空白对照及裸siRNA组相比较，大大提高了基因沉默的效率。而细胞蛋白BCA实验结果显示，与空白对照PBS与siRNA组相比，TMEA脂质体包裹siRNA制剂蛋白BCA水平相近，并未造成细胞毒性。It can be seen from the results that the liposome-encapsulated siRNA prepared by the TMEA amphiphilic compound entered the cell and specifically silenced the target gene. Compared with the PBS blank control and naked siRNA group, the efficiency of gene silencing was greatly improved . The results of the cell protein BCA experiment showed that, compared with the blank control PBS and siRNA groups, the protein BCA level of the TMEA liposome-encapsulated siRNA preparation was similar, and did not cause cytotoxicity.
表7Table 7
实施例11、T1脂质体/基因复合物人肺癌H1299-pGL3细胞转染效果Example 11, T1 liposome/gene complex human lung cancer H1299-pGL3 cell transfection effect
本实施例利用T1脂质制备的脂质体/luciferase-siRNA复合物对人非小细胞肺癌H1299细胞进行细胞转染，由其基因沉默效果，来观察载体脂质对基因药物的转运情况。In this example, the liposome/luciferase-siRNA complex prepared from T1 lipid was used to transfect human non-small cell lung cancer H1299 cells, and the gene silencing effect was used to observe the transfer of the carrier lipid to the gene drug.
1.脂质/基因复合物的制备：1. Preparation of lipid/gene complexes:
T1/基因复合物制备方法如实施例8，以T1/DPPC/Chol-(40/20/40mol)摩尔比制备脂质体，按照N/P-20/1包裹荧光素酶luciferase-siRNA，制备脂质/基因复合物；T1/gene complex preparation method as in Example 8, prepare liposomes with T1/DPPC/Chol-(40/20/40mol) molar ratio, wrap luciferase luciferase-siRNA according to N/P-20/1, prepare lipid/gene complexes;
2.H1299细胞收集与培养方法如实施例10；2. The method for collecting and culturing H1299 cells is as in Example 10;
3.复合物与细胞孵育及测定方法如实施例10；3. The incubation and assay methods of the complex and cells are as in Example 10;
效果验证：Effect verification:
(1)T1/luciferase-siRNA复合物粒径分布及均一度结果如表8所示；(1) The particle size distribution and uniformity of the T1/luciferase-siRNA complex are shown in Table 8;
(2)T1/luciferase-siRNA复合物转染H1299-pGL3细胞，基因沉默结果如图12所示；(2) T1/luciferase-siRNA complex was transfected into H1299-pGL3 cells, and the gene silencing results are shown in Figure 12;
(3)T1/luciferase-siRNA复合物转染H1299-pGL3细胞，BCA蛋白结果如图13所示；(3) T1/luciferase-siRNA complex was transfected into H1299-pGL3 cells, and the results of BCA protein were shown in Figure 13;
由结果中可以看出，经T1脂质制备的脂质体包裹siRNA进入细胞后，特异性沉默了目的基因，与PBS空白对照及裸siRNA组相比较，大大提高了基因沉默的效率。It can be seen from the results that after the liposome-wrapped siRNA prepared by T1 lipid entered the cells, the target gene was specifically silenced. Compared with the PBS blank control and naked siRNA group, the efficiency of gene silencing was greatly improved.
而细胞蛋白BCA实验结果显示，与空白对照PBS与siRNA组相比，TMEA脂质体包裹siRNA制剂蛋白BCA水平相近，并未造成细胞毒性。The results of the cell protein BCA experiment showed that, compared with the blank control PBS and siRNA groups, the protein BCA level of the TMEA liposome-encapsulated siRNA preparation was similar, and did not cause cytotoxicity.
表8Table 8
实施例12、T2/基因复合物人肺癌H1299-pGL3细胞转染效果Example 12, T2/gene complex human lung cancer H1299-pGL3 cell transfection effect
本实施例利用T2两亲性化合物制备的脂质体/luciferase-siRNA复合物对人非小细胞肺癌H1299细胞进行细胞转染，由其基因沉默效果，来观察载体脂质对基因药物的转运情况。In this example, the liposome/luciferase-siRNA complex prepared by the T2 amphiphilic compound was used to transfect human non-small cell lung cancer H1299 cells, and the gene silencing effect was used to observe the transfer of the carrier lipid to the gene drug .
1.脂质/基因复合物的制备：1. Preparation of lipid/gene complexes:
T2脂质/基因复合物制备方法如实施例9，以T2/DPPC/Chol-(40/30/40mol)摩尔比制备脂质体，按照N/P-20/1包裹荧光素酶luciferase-siRNA，制备脂质/基因复合物；T2 lipid/gene complex preparation method as in Example 9, prepare liposomes with T2/DPPC/Chol-(40/30/40mol) molar ratio, and wrap luciferase luciferase-siRNA according to N/P-20/1 , preparation of lipid/gene complexes;
2.H1299细胞收集与培养方法如实施例10；2. The method for collecting and culturing H1299 cells is as in Example 10;
3.复合物与细胞孵育及测定方法如实施例10；3. The incubation and assay methods of the complex and cells are as in Example 10;
效果验证：Effect verification:
(1)T2脂质/luciferase-siRNA复合物粒径分布及均一度结果如表9所示；(1) The particle size distribution and uniformity of the T2 lipid/luciferase-siRNA complex are shown in Table 9;
(2)T2脂质/luciferase-siRNA复合物转染H1299-pGL3细胞，基因沉默结果如图14所示；(2) T2 lipid/luciferase-siRNA complex was transfected into H1299-pGL3 cells, and the gene silencing results are shown in Figure 14;
(3)T2脂质/luciferase-siRNA复合物转染H1299-pGL3细胞，BCA蛋白结果如图15所示；(3) T2 lipid/luciferase-siRNA complex was transfected into H1299-pGL3 cells, and the results of BCA protein were shown in Figure 15;
由结果中可以看出，经T2脂质制备的脂质体包裹siRNA进入细胞后，特异性沉默了目的基因，与PBS空白对照及裸siRNA组相比较，大大提高了基因沉默的效率。It can be seen from the results that after the liposome-wrapped siRNA prepared by T2 lipid enters the cells, the target gene is specifically silenced. Compared with the PBS blank control and naked siRNA group, the efficiency of gene silencing is greatly improved.
而细胞蛋白BCA实验结果显示，与空白对照PBS与siRNA组相比，TMEA脂质体包裹siRNA制剂蛋白BCA水平相近，并未造成细胞毒性。The results of the cell protein BCA experiment showed that, compared with the blank control PBS and siRNA groups, the protein BCA level of the TMEA liposome-encapsulated siRNA preparation was similar, and did not cause cytotoxicity.
表9Table 9
实施例16、组织切片后共聚焦显微镜观察TMEA/Cy-5-siRNA复合物在肝内分布Example 16. Observation of the distribution of TMEA/Cy-5-siRNA complex in the liver by confocal microscope after tissue sectioning
本实施例利用TMEA两亲性衍生物制备的脂质体包裹Cy-5-siRNA制备的脂质/siRNA复合物，动物给药后，肝脏组织切片后，共聚焦显微镜下观察TMEA脂质/siRNA复合物分布，从而探知载体脂质对基因药物的转运情况。In this example, the lipid/siRNA complex prepared by encapsulating Cy-5-siRNA in liposomes prepared by amphiphilic derivatives of TMEA was used. After administration to animals, liver tissue slices were used to observe TMEA lipid/siRNA under a confocal microscope. Complex distribution, so as to explore the transfer of carrier lipids to gene drugs.
1.TMEA脂质/Cy5-siRNA复合物制备：TMEA脂质/Cy5-siRNA复合物制备方法及比例如实施例7；1. Preparation of TMEA lipid/Cy5-siRNA complex: the preparation method and ratio of TMEA lipid/Cy5-siRNA complex are shown in Example 7;
2.组织切片2. Tissue sectioning
(1)取材：取成年健康ICR小鼠20g，尾静脉注射TMEA脂质/Cy5-siRNA复合物200μL，约10μg Cy5-siRNA，4h后，颈椎脱臼，解剖，取出肝脏；(1) Material collection: Take 20 g of adult healthy ICR mice, inject 200 μL of TMEA lipid/Cy5-siRNA complex into the tail vein, and about 10 μg of Cy5-siRNA, after 4 hours, the cervical vertebrae are dislocated, dissected, and the liver is removed;
(2)速冻：将取出的肝脏修成小块，置于OCT中包埋，迅速置于液氮中速冻，成块后立即转移至冰冻切片机，准备切片；(2) Quick-freezing: the removed liver was trimmed into small pieces, embedded in OCT, quickly frozen in liquid nitrogen, and transferred to a cryostat immediately after forming a piece, ready to be sliced;
(3)切片：将冰冻切片机恒温箱调至-25度，将包埋块沿切片方向修整成长方形或正方形，并将修整后的组织小块放入标本盘中，切片厚度调至20μm,连续切片，直接用预处理后的载玻片粘片，并放入多聚甲醛中固定5min；(3) Sectioning: Adjust the incubator of the cryostat to -25 degrees, trim the embedding block into a rectangle or square along the sectioning direction, put the trimmed tissue pieces into the specimen tray, and adjust the section thickness to 20 μm. For serial sections, stick the slides directly with pretreated slides, and fix them in paraformaldehyde for 5 minutes;
(4)漂洗：将固定后的切片转移至装有1xPBS的染色缸中，漂洗3min,漂洗3次；(4) Rinsing: transfer the fixed sections to a staining jar filled with 1xPBS, rinse for 3 min, and rinse 3 times;
(5)封闭:将漂洗后的切片用擦镜纸擦干组织周围的水分，用免疫组化笔在组织周围画圈，加入5％的Donkey serum,于湿盒内37摄氏度封闭30min；(5) Sealing: dry the rinsed sections with lens tissue to dry the moisture around the tissue, draw a circle around the tissue with an immunohistochemical pen, add 5% Donkey serum, and seal in a wet box at 37 degrees Celsius for 30 minutes;
(6)一抗孵育：吸收封闭液，立即加入稀释好的一抗，于湿盒内37度封闭12-16h；(6) Primary antibody incubation: absorb the blocking solution, immediately add the diluted primary antibody, and block in a humid box at 37 degrees for 12-16 hours;
(7)二抗孵育：吸掉一抗，加入稀释好的二抗，于湿盒内37度孵育约30min；(7) Secondary antibody incubation: absorb the primary antibody, add the diluted secondary antibody, and incubate at 37°C for about 30 minutes in a wet box;
(8)DAPI染色：吸掉二抗稀释液，加入DAPI,于湿盒内37摄氏度孵育约30min；(8) DAPI staining: absorb the secondary antibody dilution, add DAPI, and incubate in a humid box at 37 degrees Celsius for about 30 minutes;
(9)封片：用Vector公司防荧光淬灭封片剂封片，并用指甲油封闭盖玻片四周。(9) Mount the slide: use Vector's anti-fluorescence quenching mountant to seal the slide, and seal the coverslip around with nail polish.
3.荧光共聚焦显微镜(Confocal Microscopy)信号采集3. Fluorescence confocal microscopy (Confocal Microscopy) signal acquisition
(1)DAPI扫描序列：(Excitation：405nm，Emission：419-460nm)；(1) DAPI scanning sequence: (Excitation: 405nm, Emission: 419-460nm);
(2)Alexa Fluor488扫描序列：(Excitation：500-550nm)；(2) Alexa Fluor488 scanning sequence: (Excitation: 500-550nm);
(3)Dylight549扫描序列：(Excitation：561nm，Emission：559-610nm)；(3) Dylight549 scanning sequence: (Excitation: 561nm, Emission: 559-610nm);
(4)Cy5-siRNA扫描序列：(Excitation：633nm，Emission：650-750nm)。(4) Cy5-siRNA scanning sequence: (Excitation: 633nm, Emission: 650-750nm).
效果验证：Effect verification:
表10Table 10
TMEA脂质/Cy5-siRNA复合物组织切片后共聚焦显微镜观察复合物在肝内分布，如图16所示；蓝色信号为DAPI染色的细胞核，星形绿色信号为kupffer细胞，红色信号为TMEA脂质/Cy5-siRNA。由图15可以看出，在体内给药4小时后，大量TMEA阳离子脂质体包裹的Cy5-siRNA红色信号分布在肝脏细胞内。如果复合物红色信号被巨噬细胞kupffer细胞绿色信号吞噬，会呈黄色。因此从图16可以看出，少数Cy5-siRNA被巨噬细胞吞噬，更多的Cy5-siRNA信号聚集到了肝实质细胞内。说明TMEA脂质载体系统能将Cy5-siRNA体内安全高效递送到肝脏，更可贵的是，将siRNA更多递送到了肝实质细胞中，对于治疗肝脏疾病有非常大的优势。The distribution of the TMEA lipid/Cy5-siRNA complex in the liver was observed by confocal microscopy after tissue sectioning, as shown in Figure 16; the blue signal is DAPI-stained nuclei, the star-shaped green signal is kupffer cells, and the red signal is TMEA Lipid/Cy5-siRNA. It can be seen from Figure 15 that after 4 hours of in vivo administration, the red signal of Cy5-siRNA encapsulated in a large amount of TMEA cationic liposomes was distributed in the liver cells. If the complex red signal is engulfed by the macrophage kupffer cell green signal, it will appear yellow. Therefore, it can be seen from FIG. 16 that a small amount of Cy5-siRNA was phagocytized by macrophages, and more Cy5-siRNA signals were gathered in hepatic parenchymal cells. It shows that the TMEA lipid carrier system can safely and efficiently deliver Cy5-siRNA to the liver in vivo. What's more valuable is that more siRNA is delivered to the liver parenchymal cells, which has great advantages for the treatment of liver diseases.
实施例17、组织切片后共聚焦显微镜观察T1脂质/Cy-5-siRNA复合物在肝内分布Example 17. Observation of the distribution of T1 lipid/Cy-5-siRNA complex in the liver by confocal microscope after tissue sectioning
本实施例利用T1阳离子脂质包裹Cy-5-siRNA制备的脂质/siRNA复合物，动物给药后，肝脏组织切片后，共聚焦显微镜下观察TMEA脂质/siRNA复合物分布，从而探知载体脂质对基因药物的转运情况。In this example, the lipid/siRNA complex prepared by encapsulating Cy-5-siRNA with T1 cationic lipid was used. After administration to animals, the distribution of TMEA lipid/siRNA complex was observed under a confocal microscope after the liver tissue was sliced, so as to detect the carrier Lipid transfer of gene drugs.
1.T1脂质/Cy5-siRNA复合物制备：T1脂质/Cy5-siRNA复合物制备方法及比例如实施例8；1. Preparation of T1 lipid/Cy5-siRNA complex: the preparation method and ratio of T1 lipid/Cy5-siRNA complex are shown in Example 8;
2.组织切片方法如实施例16；2. The tissue sectioning method is as in Example 16;
3.共聚焦显微镜Confocal(Leica TS SP8)观察实验方法如实施例16。3. Confocal (Leica TS SP8) observation The experimental method is as in Example 16.
效果验证：Effect verification:
T1脂质/Cy5-siRNA复合物组织切片后共聚焦显微镜观察复合物在肝内分布，如图17所示；蓝色信号为DAPI染色的细胞核，星形绿色信号为kupffer细胞，红色信号为T1脂质/Cy5-siRNA。由图可以看出，在体内给药4小时后，大量T1阳离子脂质体包裹的Cy5-siRNA红色信号分布在肝脏细胞内。如果复合物红色信号被巨噬细胞kupffer细胞绿色信号吞噬，会呈黄色。因此从图17可以看出，少数Cy5-siRNA被巨噬细胞吞噬，更多的Cy5-siRNA信号聚集到了肝实质细胞内。说明T1脂质载体系统能将Cy5-siRNA体内安全高效递送到肝脏，更可贵的是，将siRNA更多递送到了肝实质细胞中，对于治疗肝脏疾病有非常大的优势。The distribution of the T1 lipid/Cy5-siRNA complex in the liver was observed by confocal microscopy after tissue sectioning, as shown in Figure 17; the blue signal is DAPI-stained nuclei, the star-shaped green signal is kupffer cells, and the red signal is T1 Lipid/Cy5-siRNA. It can be seen from the figure that after 4 hours of in vivo administration, the red signal of Cy5-siRNA encapsulated in a large number of T1 cationic liposomes is distributed in the liver cells. If the complex red signal is engulfed by the macrophage kupffer cell green signal, it will appear yellow. Therefore, it can be seen from FIG. 17 that a small amount of Cy5-siRNA was phagocytized by macrophages, and more Cy5-siRNA signals were gathered in hepatic parenchymal cells. It shows that the T1 lipid carrier system can safely and efficiently deliver Cy5-siRNA to the liver in vivo, and what's more, it can deliver more siRNA to liver parenchymal cells, which has great advantages for the treatment of liver diseases.
表11Table 11
实施例18、组织切片后共聚焦显微镜观察T2脂质/Cy-5-siRNA复合物在肝内分布Example 18. Observation of the distribution of T2 lipid/Cy-5-siRNA complex in the liver by confocal microscope after tissue sectioning
本实施例利用T2两亲性衍生物化合物制备的脂质体包裹Cy-5-siRNA制备的脂质/siRNA复合物，动物给药后，肝脏组织切片后，共聚焦显微镜下观察T2脂质/siRNA复合物分布，从而探知载体脂质对基因药物的转运情况。In this example, liposomes prepared by T2 amphiphilic derivative compounds were used to encapsulate the lipid/siRNA complex prepared by Cy-5-siRNA. After administration to animals, liver tissue sections were observed under a confocal microscope to observe the T2 lipid/siRNA complex. The distribution of siRNA complexes can be used to detect the transfer of gene drugs to carrier lipids.
1.T2脂质/Cy5-siRNA复合物制备：T2脂质/Cy5-siRNA复合物制备方法及比例如实施例8；1. Preparation of T2 lipid/Cy5-siRNA complex: The preparation method and ratio of T2 lipid/Cy5-siRNA complex are shown in Example 8;
2.组织切片方法如实施例16；2. The tissue sectioning method is as in Example 16;
3.共聚焦显微镜Confocal(Leica TS SP8)观察实验方法如实施例16。3. Confocal (Leica TS SP8) observation The experimental method is as in Example 16.
效果验证：Effect verification:
T2脂质/Cy5-siRNA复合物组织切片后共聚焦显微镜观察复合物在肝内分布，如图18所示；蓝色信号为DAPI染色的细胞核，星形绿色信号为kupffer细胞，红色信号为T2脂质/Cy5-siRNA。由图可以看出，在体内给药4小时后，大量T2阳离子脂质体包裹的Cy5-siRNA红色信号分布在肝脏细胞内。如果复合物红色信号被巨噬细胞kupffer细胞绿色信号吞噬，会呈黄色。因此从图18可以看出，少数Cy5-siRNA被巨噬细胞吞噬，更多的Cy5-siRNA信号聚集到了肝实质细胞内。说明T2脂质载体系统能将Cy5-siRNA体内安全高效递送到肝脏，更可贵的是，将siRNA更多递送到了肝实质细胞中，对于治疗肝脏疾病有非常大的优势。The distribution of the T2 lipid/Cy5-siRNA complex in the liver was observed by confocal microscopy after tissue sectioning, as shown in Figure 18; the blue signal is DAPI-stained nuclei, the star-shaped green signal is kupffer cells, and the red signal is T2 Lipid/Cy5-siRNA. It can be seen from the figure that after 4 hours of in vivo administration, the red signal of Cy5-siRNA encapsulated in a large number of T2 cationic liposomes is distributed in the liver cells. If the complex red signal is engulfed by the macrophage kupffer cell green signal, it will appear yellow. Therefore, it can be seen from FIG. 18 that a small amount of Cy5-siRNA was phagocytized by macrophages, and more Cy5-siRNA signals were gathered in hepatic parenchymal cells. It shows that the T2 lipid carrier system can safely and efficiently deliver Cy5-siRNA to the liver in vivo, and what's more, it can deliver more siRNA to liver parenchymal cells, which has great advantages for the treatment of liver diseases.
表12Table 12
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.
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| CN201410222444.XACN105085292B (en) | 2014-05-23 | 2014-05-23 | Amphipathic derivatives of 3 ((2 (dimethylamino) ethyl group) (methyl) amino) propionic acid and application thereof |
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