【技术领域】【Technical field】
本发明涉及靶向基因传递技术领域，特别是一种靶向传递小干扰RNA的超分子组装体及制备方法。The invention relates to the technical field of targeted gene delivery, in particular to a supramolecular assembly and a preparation method for targeted delivery of small interfering RNA.
【背景技术】【Background technique】
当今社会由于雾霾、环境污染、电离辐射、食品安全以及遗传等因素的影响，癌症的发病率正在逐年上升，严重威胁到了人类的健康。为此，手术治疗、化学治疗和放射治疗作为临床治疗癌症的主要手段得以广泛应用。然而，这些疗法由于治疗效果不佳、易复发以及严重的毒副作用等使得癌症至今无法被人类所攻克。因此，化学家、材料学家和生物医学工作者开发了各种药物传递体系如脂质体、无机纳米粒子、囊泡以及碳纳米材料等用以增强药物传递体系的生物兼容性和靶向性，以期待增强治疗效果并降低毒副作用。然而，这些疗法都是治标不治本的，要想彻底根治癌症，就要从癌症发病的源头出发对其加以遏制。近几年出现的基因治疗为攻克癌症打开了一扇大门。其原理是将具有生物功能的核酸(如质粒和线性双链DNA，或者小干扰RNA(siRNA)等)转移或运送到癌细胞中，修复损伤的基因或者抑制细胞内异常基因的表达，继而诱导不凋亡的癌细胞发生凋亡，从而达到治愈癌症的目的。然而基因转染的低效率成为了制约该方法广泛应用的瓶颈。由于潜在的安全性问题以及昂贵的成本，病毒载体并没有得到广泛的发展，因此设计与构筑具有生物兼容性和可控释放功能并且具备高的基因转染效率的靶向基因传递体系成为了前沿的研究热点。此外，非共价相互作用载体由于其优良的缓释效果、较强的键合能力以及较低的毒副作用等优点，在新近出现的药物/基因载体上广为应用。In today's society, due to the influence of smog, environmental pollution, ionizing radiation, food safety, genetics and other factors, the incidence of cancer is increasing year by year, seriously threatening human health. For this reason, surgical treatment, chemotherapy and radiotherapy are widely used as the main means of clinical treatment of cancer. However, due to the poor therapeutic effect, easy recurrence and serious side effects of these therapies, cancer cannot be conquered by humans so far. Therefore, chemists, material scientists and biomedical workers have developed various drug delivery systems such as liposomes, inorganic nanoparticles, vesicles and carbon nanomaterials to enhance the biocompatibility and targeting of drug delivery systems , in order to enhance the therapeutic effect and reduce the side effects. However, these treatments are all palliatives, not the root cause. To completely cure cancer, it is necessary to curb it from the source of cancer. In recent years, gene therapy has opened a door for conquering cancer. The principle is to transfer or transport nucleic acids with biological functions (such as plasmids and linear double-stranded DNA, or small interfering RNA (siRNA), etc.) into cancer cells, repair damaged genes or inhibit the expression of abnormal genes in cells, and then induce Non-apoptotic cancer cells undergo apoptosis, thereby achieving the purpose of curing cancer. However, the low efficiency of gene transfection has become a bottleneck restricting the wide application of this method. Due to potential safety issues and high costs, viral vectors have not been widely developed, so the design and construction of targeted gene delivery systems with biocompatibility and controlled release functions and high gene transfection efficiency have become the forefront research hotspots. In addition, non-covalent interaction carriers are widely used in newly emerging drug/gene carriers due to their excellent sustained release effect, strong bonding ability, and low toxic and side effects.
【发明内容】【Content of invention】
本发明的目的是针对上述技术分析和存在问题，提供一种靶向传递小干扰RNA的超分子组装体及制备方法，该超分子组装体能够有效沉默癌细胞内特定内源和外源基因的表达，且其毒副作用很低，制备方法简单且产率较高，适于放大合成和实际生产应用。The purpose of the present invention is to provide a supramolecular assembly and a preparation method for the targeted delivery of small interfering RNA in view of the above-mentioned technical analysis and existing problems. The supramolecular assembly can effectively silence specific endogenous and exogenous genes in cancer cells. expression, and its toxic and side effects are very low, the preparation method is simple and the yield is high, and it is suitable for scale-up synthesis and practical production application.
本发明的技术方案：Technical scheme of the present invention:
一种靶向传递小干扰RNA的超分子组装体，为基于β-环糊精修饰透明质酸、金刚烷多胺化合物、葫芦脲[6]以及小干扰RNA合成的四元超分子组装体，其中β-环糊精修饰透明质酸分子式为(C14H21NO11)98(C58H95N3O44)17,平均一条高分子链上修饰17个环糊精单元，金刚烷多胺化合物化学分子式为C32H56Br4N6O4；该四元超分子组装体以β-环糊精与金刚烷以及金刚烷多胺链与葫芦脲[6]间强的非共价相互作用，和金刚烷多胺链与小干扰RNA间静电相互作用为基础，形成以亲水的透明质酸为外壳的超分子纳米粒子，纳米粒子粒径为30-40nm。A supramolecular assembly for targeted delivery of small interfering RNA, a quaternary supramolecular assembly based on β-cyclodextrin modified hyaluronic acid, adamantane polyamine compound, cucurbituril [6] and small interfering RNA synthesis, Among them, the molecular formula of β-cyclodextrin-modified hyaluronic acid is (C14 H21 NO11 )98 (C58 H95 N3 O44 )17 , and an average of 17 cyclodextrin units are modified on one polymer chain. The chemical formula of the amine compound is C32 H56 Br4 N6 O4 ; the quaternary supramolecular assembly has a strong non-covalent relationship between β-cyclodextrin and adamantane and adamantane polyamine chain and cucurbituril[6]. Based on the interaction, and the electrostatic interaction between the adamantane polyamine chain and the small interfering RNA, a supramolecular nanoparticle with a hydrophilic hyaluronic acid as the shell is formed, and the particle size of the nanoparticle is 30-40nm.
一种所述靶向传递小干扰RNA的超分子组装体的制备方法，步骤如下：A method for preparing a supramolecular assembly targeting delivery of small interfering RNA, the steps are as follows:
(1)β-环糊精修饰透明质酸的制备(1) Preparation of β-cyclodextrin modified hyaluronic acid
将分子量为46000的透明质酸钠溶于pH为7.2、浓度为1mmol/L的磷酸缓冲溶液中，再加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺，搅拌反应30分钟，得到反应液；将6-去氧-6-乙二胺-β-环糊精溶于pH为7.2的磷酸缓冲溶液中，6-去氧-6-乙二胺-β-环糊精与磷酸缓冲溶液的用量比为0.1mol/L，再加入到上述反应液中，在25℃条件下搅拌24小时，然后装入截留范围为8000-14000的透析袋中连续透析5天，将所得溶液冻干，制得β-环糊精修饰透明质酸；Dissolve sodium hyaluronate with a molecular weight of 46000 in a phosphate buffer solution with a pH of 7.2 and a concentration of 1 mmol/L, and then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, stirred and reacted for 30 minutes to obtain a reaction solution; 6-deoxy-6-ethylenediamine-β-cyclodextrin was dissolved in a phosphate buffer solution with a pH of 7.2, and 6- The ratio of deoxy-6-ethylenediamine-β-cyclodextrin to phosphate buffer solution is 0.1mol/L, and then added to the above reaction solution, stirred at 25°C for 24 hours, and then filled with a cut-off range of 8000-14000 dialysis bags were continuously dialyzed for 5 days, and the resulting solution was freeze-dried to obtain β-cyclodextrin-modified hyaluronic acid;
(2)金刚烷多胺化合物的制备(2) Preparation of adamantane polyamine compound
1)将二叠氮甲基苯酚化合物(化合物1)、金刚烷溴甲基酮和无水碳酸钾在丙酮中混合，加热至56℃回流搅拌反应10-12h，然后过滤，将滤液旋干，加入体积比为1:1的三氯甲烷和水混合液进行萃取，有机相用无水氯化镁干燥，减压蒸馏除去溶剂得到粗品，然后以石油醚和乙酸乙酯混合液为展开剂，该混合液中石油醚和乙酸乙酯的体积比为8:1，用200-300目硅胶柱进行分离，制得二叠氮甲基苯基金刚烷化合物(化合物2)的纯品；1) Mix diazide methylphenol compound (compound 1), adamantyl bromomethyl ketone and anhydrous potassium carbonate in acetone, heat to 56° C. under reflux and stir for 10-12 hours, then filter, spin the filtrate to dryness, Add a mixed solution of chloroform and water with a volume ratio of 1:1 for extraction, dry the organic phase with anhydrous magnesium chloride, distill off the solvent under reduced pressure to obtain a crude product, and then use a mixed solution of petroleum ether and ethyl acetate as a developing solvent. The volume ratio of petroleum ether and ethyl acetate in the liquid is 8:1, and the silica gel column of 200-300 mesh is used for separation, and the pure product of diazidemethylphenyladamantane compound (compound 2) is obtained;
2)将二叠氮甲基苯基金刚烷化合物(化合物2)和三苯基膦溶于无水四氢呋喃中，在室温条件下搅拌12小时，然后减压蒸馏除去溶剂，将所得固体溶于乙酸乙酯中，加入浓度为36.5wt％的盐酸后得到白色沉淀，收集沉淀并用乙酸乙酯洗涤，得到二胺甲基苯基金刚烷化合物(化合物3)的纯品；2) Dissolve the diazidemethylphenyladamantane compound (compound 2) and triphenylphosphine in anhydrous tetrahydrofuran, stir at room temperature for 12 hours, then distill off the solvent under reduced pressure, and dissolve the resulting solid in acetic acid In ethyl ester, after adding hydrochloric acid with a concentration of 36.5wt%, a white precipitate was obtained, and the precipitate was collected and washed with ethyl acetate to obtain a pure product of diaminomethylphenyladamantane compound (compound 3);
3)将二胺甲基苯基金刚烷化合物(化合物3)、氯甲酸苄酯保护的五氟苯酚多胺化合物(化合物4)和三乙胺溶于二氯甲烷中，室温下搅拌反应20小时，减压蒸馏除去溶剂，加入体积比为1:1的二氯甲烷和水混合液进行萃取，有机相用无水硫酸镁干燥，然后减压蒸馏除去溶剂得到粗品，以乙酸乙酯和石油醚混合液为展开剂，该混合液中石油醚和乙酸乙酯的容积比依次为1:2变至4:1变至6:1最后为纯乙酸乙酯，用200-300目硅胶柱进行分离，制得氯甲酸苄酯保护的金刚烷多胺化合物(化合物5)的纯品；3) Diaminomethylphenyladamantane compound (compound 3), pentafluorophenol polyamine compound (compound 4) protected by benzyl chloroformate and triethylamine were dissolved in dichloromethane, stirred and reacted at room temperature for 20 hours , remove the solvent by distillation under reduced pressure, add dichloromethane and water mixed solution with a volume ratio of 1:1 for extraction, dry the organic phase with anhydrous magnesium sulfate, then remove the solvent by distillation under reduced pressure to obtain the crude product, and use ethyl acetate and petroleum ether The mixed solution is a developer, and the volume ratio of petroleum ether and ethyl acetate in the mixed solution is sequentially changed from 1:2 to 4:1 to 6:1, and finally pure ethyl acetate is separated by a 200-300 mesh silica gel column. Obtain the pure product of the adamantane polyamine compound (compound 5) of benzyl chloroformate protection;
4)将氯甲酸苄酯保护的金刚烷多胺化合物(化合物5)溶于氢溴酸与乙酸的混合溶液中，氢溴酸在与乙酸的混合溶液中的浓度为33wt％，室温搅拌反应10小时，加入过量的乙醚产生白色沉淀，离心并用乙醚进行洗涤即可制得金刚烷多胺化合物的纯品；4) The adamantane polyamine compound (compound 5) protected by benzyl chloroformate was dissolved in a mixed solution of hydrobromic acid and acetic acid, the concentration of hydrobromic acid in the mixed solution with acetic acid was 33 wt%, and the reaction was stirred at room temperature for 10 hour, add excessive ether to produce white precipitate, centrifuge and wash with ether to obtain the pure product of adamantane polyamine compound;
(3)靶向传递小干扰RNA的超分子组装体的制备(3) Preparation of supramolecular assemblies for targeted delivery of small interfering RNA
将β-环糊精修饰透明质酸、金刚烷多胺化合物和葫芦脲[6]混合并溶于水中，进行超声溶解，然后加入浓度为20μmol/L的小干扰RNA的水溶液，用200μL移液枪反复吹打20次，制得靶向传递小干扰RNA的四元超分子组装体。Mix β-cyclodextrin-modified hyaluronic acid, adamantane polyamine compound and cucurbituril [6] and dissolve in water, perform ultrasonic dissolution, then add an aqueous solution of small interfering RNA with a concentration of 20 μmol/L, pipette with 200 μL The gun was blown repeatedly 20 times to prepare a quaternary supramolecular assembly for targeted delivery of small interfering RNA.
所述步骤(1)中透明质酸钠与磷酸缓冲溶液的用量比为0.073mmol/L，透明质酸钠、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、N-羟基硫代琥珀酰亚胺和6-去氧-6-乙二胺-β-环糊精的摩尔比为0.0044:1.75:1.75:1。In described step (1), the consumption ratio of sodium hyaluronate and phosphate buffer solution is 0.073mmol/L, sodium hyaluronate, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride The molar ratio of salt, N-hydroxysulfosuccinimide and 6-deoxy-6-ethylenediamine-β-cyclodextrin was 0.0044:1.75:1.75:1.
所述步骤(2)的1)中二叠氮甲基苯酚化合物(化合物1)与丙酮的用量比为0.026mol/L，二叠氮甲基苯酚化合物(化合物1)与金刚烷溴甲基酮和无水碳酸钾的摩尔比是1:0.74:3。In 1) of the step (2), the amount ratio of diazide methylphenol compound (compound 1) to acetone is 0.026mol/L, diazide methylphenol compound (compound 1) and adamantyl bromomethyl ketone The molar ratio to anhydrous potassium carbonate is 1:0.74:3.
所述步骤(2)的2)中二叠氮甲基苯基金刚烷化合物(化合物2)与无水四氢呋喃的用量比为0.097mol/L，二叠氮甲基苯基金刚烷化合物(化合物2)与三苯基膦的摩尔比为1:3，无水四氢呋喃与浓度为36.5wt％的盐酸的体积比为40:1。In 2) of the step (2), the amount ratio of the diazidemethylphenyladamantane compound (compound 2) to anhydrous tetrahydrofuran is 0.097mol/L, and the diazidemethylphenyladamantane compound (compound 2 ) to triphenylphosphine in a molar ratio of 1:3, and the volume ratio of anhydrous tetrahydrofuran to hydrochloric acid with a concentration of 36.5wt% was 40:1.
所述步骤(2)的3)中二胺甲基苯基金刚烷化合物(化合物3)与二氯甲烷的用量比为0.015mol/L，二胺甲基苯基金刚烷化合物(化合物3)与氯甲酸苄酯保护的五氟苯酚多胺化合物(化合物4)和三乙胺的摩尔比为1:2.5:4。In 3) of the step (2), the amount ratio of the diaminomethylphenyladamantane compound (compound 3) to methylene chloride is 0.015mol/L, and the diaminomethylphenyladamantane compound (compound 3) and The molar ratio of benzyl chloroformate-protected pentafluorophenol polyamine compound (compound 4) to triethylamine is 1:2.5:4.
所述步骤(2)的4)中氯甲酸苄酯保护的金刚烷多胺化合物(化合物5)与氢溴酸-乙酸的混合溶液的用量比为0.04mol/L。The dosage ratio of the mixed solution of the benzyl chloroformate-protected adamantane polyamine compound (compound 5) and hydrobromic acid-acetic acid in 4) of the step (2) is 0.04mol/L.
所述步骤(3)中β-环糊精修饰透明质酸与水的用量比为2.0×10-4mol/L，β-环糊精修饰透明质酸、金刚烷多胺化合物和葫芦脲[6]的摩尔比为1:17:34，β-环糊精修饰透明质酸/金刚烷多胺化合物/葫芦脲[6]混合溶液与小干扰RNA水溶液的体积比为1:1。In the step (3), the dosage ratio of β-cyclodextrin modified hyaluronic acid to water is 2.0×10-4 mol/L, β-cyclodextrin modified hyaluronic acid, adamantane polyamine compound and cucurbituril[ 6] molar ratio is 1:17:34, the volume ratio of β-cyclodextrin modified hyaluronic acid/adamantane polyamine compound/cucurbituril [6] mixed solution and small interfering RNA aqueous solution is 1:1.
本发明的优点是：该靶向传递小干扰RNA的四元超分子组装体，葫芦脲[6]与多胺链的键合使得多胺链的氮原子pKa值发生改变，使得多胺链的电正性大大增强，能够强烈地与带负电的小干扰RNA进行键合，避免小干扰RNA由于水解和与RNA分解酶作用而分解失活；此外，该四元超分子组装体的透明质酸外壳能够与癌细胞表面过量表达的透明质酸受体进行特异性键合作用，然后四元超分子组装体通过细胞的内吞作用特异性地进入到癌细胞当中，再通过癌细胞内过量表达的透明质酸酶的水解作用使得四元组装体能够得到有效降解从而使小干扰RNA得到释放，大大提高了细胞的基因转染效率，使得细胞内内源和外源性的特定基因得到有效沉默；该组装体靶向性好，细胞毒副作用很低，转染效率较高，制备方法较为方便，在未来癌症的基因疗法上具有较好的应用前景。The advantage of the present invention is that: the quaternary supramolecular assembly targeting delivery of small interfering RNA, the bonding of cucurbituril [6] to the polyamine chain makes the pKa value of the nitrogen atom of the polyamine chain change, making the polyamine chain The electropositiveness is greatly enhanced, and it can strongly bond with the negatively charged small interfering RNA, avoiding the decomposition and inactivation of the small interfering RNA due to hydrolysis and the action of RNA decomposing enzymes; in addition, the hyaluronic acid of the quaternary supramolecular assembly The shell can specifically bond with the overexpressed hyaluronic acid receptors on the surface of cancer cells, and then the quaternary supramolecular assembly specifically enters the cancer cells through the endocytosis of the cells, and then through the overexpression in the cancer cells The hydrolysis of hyaluronidase can effectively degrade the quadruple assembly and release small interfering RNA, which greatly improves the gene transfection efficiency of cells and effectively silences endogenous and exogenous specific genes in cells The assembly has good targeting performance, low cytotoxicity and side effects, high transfection efficiency and convenient preparation method, and has a good application prospect in future cancer gene therapy.
【附图说明】【Description of drawings】
图1为金刚烷多胺化合物的合成路线图。Figure 1 is a synthetic route diagram of adamantane polyamine compounds.
图2为该四元超分子组装体的构筑路线示意图。Figure 2 is a schematic diagram of the construction route of the quaternary supramolecular assembly.
图3为该四元超分子组装体的透射电子显微镜图。Figure 3 is a transmission electron microscope image of the quaternary supramolecular assembly.
图4为该四元超分子组装体与小干扰RNA键合的电泳图。Fig. 4 is an electrophoresis diagram of the binding of the quaternary supramolecular assembly to the small interfering RNA.
图5为该四元超分子组装体的细胞毒性实验结果。Figure 5 shows the cytotoxicity test results of the quaternary supramolecular assembly.
图6为该四元超分子组装体对细胞外源性绿色荧光蛋白基因沉默效果图。Fig. 6 is a graph showing the effect of the quaternary supramolecular assembly on the gene silencing of exogenous green fluorescent protein in cells.
【具体实施方式】【detailed description】
下面通过实例对本发明做进一步的说明：Below by example the present invention will be further described:
实施例：Example:
一种靶向传递小干扰RNA的超分子组装体，为基于β-环糊精修饰透明质酸、金刚烷多胺化合物、葫芦脲[6]以及小干扰RNA合成的四元超分子组装体，其中β-环糊精修饰透明质酸分子式为(C14H21NO11)98(C58H95N3O44)17,平均一条高分子链上修饰17个环糊精单元，金刚烷多胺化合物化学分子式为C32H56Br4N6O4；该四元超分子组装体以β-环糊精与金刚烷以及金刚烷多胺链与葫芦脲[6]间强的非共价相互作用，和金刚烷多胺链与小干扰RNA间静电相互作用为基础，形成以亲水的透明质酸为外壳的超分子纳米粒子，纳米粒子粒径为30-40nm。A supramolecular assembly for targeted delivery of small interfering RNA, a quaternary supramolecular assembly based on β-cyclodextrin modified hyaluronic acid, adamantane polyamine compound, cucurbituril [6] and small interfering RNA synthesis, Among them, the molecular formula of β-cyclodextrin-modified hyaluronic acid is (C14 H21 NO11 )98 (C58 H95 N3 O44 )17 , and an average of 17 cyclodextrin units are modified on one polymer chain. The chemical formula of the amine compound is C32 H56 Br4 N6 O4 ; the quaternary supramolecular assembly has a strong non-covalent relationship between β-cyclodextrin and adamantane and adamantane polyamine chain and cucurbituril[6]. Based on the interaction, and the electrostatic interaction between the adamantane polyamine chain and the small interfering RNA, a supramolecular nanoparticle with a hydrophilic hyaluronic acid as the shell is formed, and the particle size of the nanoparticle is 30-40nm.
一种所述靶向传递小干扰RNA的超分子组装体的制备方法，步骤如下：A method for preparing a supramolecular assembly targeting delivery of small interfering RNA, the steps are as follows:
(1)β-环糊精修饰透明质酸的制备(1) Preparation of β-cyclodextrin modified hyaluronic acid
将100mg(0.0022mmol)分子量为46000的透明质酸钠溶于30mL pH为7.2、浓度为1mmol/L的磷酸缓冲溶液中，再加入167.7mg(0.875mmol)1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和190mg(0.875mmol)N-羟基硫代琥珀酰亚胺，搅拌反应30分钟，得到反应液；将588.5mg(0.5mmol)6-去氧-6-乙二胺-β-环糊精溶于5mL pH为7.2的磷酸缓冲溶液中，再加入到上述反应液中，在25℃条件下搅拌24小时，然后装入截留范围为8000-14000的透析袋中连续透析5天，将所得溶液冻干，制得环糊精修饰透明质酸(HACD)。Dissolve 100 mg (0.0022 mmol) of sodium hyaluronate with a molecular weight of 46000 in 30 mL of phosphate buffer solution with a pH of 7.2 and a concentration of 1 mmol/L, and then add 167.7 mg (0.875 mmol) of 1-ethyl-(3-dimethyl Aminopropyl) carbodiimide hydrochloride and 190mg (0.875mmol) N-hydroxyl sulfosuccinimide, stirred and reacted for 30 minutes to obtain a reaction solution; 588.5mg (0.5mmol) 6-deoxy-6 -Ethylenediamine-β-cyclodextrin was dissolved in 5 mL of phosphate buffer solution with a pH of 7.2, then added to the above reaction solution, stirred at 25°C for 24 hours, and then loaded into a dialyzer with a cut-off range of 8000-14000 The bag was dialyzed continuously for 5 days, and the resulting solution was freeze-dried to prepare cyclodextrin-modified hyaluronic acid (HACD).
检测显示本发明制备的β-环糊精修饰透明质酸的核磁表征如下：1H NMR(400MHz,D2O,TMS,ppm):δ1.97(s，3H，H of methyl group of HA)，2.84-3.93(m，16.24H，H of HAand C-3,C-5,C-6,C-2,C-4ofβ-CD)，4.45-4.50(m，2H，H of HA)，5.04-5.10(m，1.06H，H ofC-1ofβ-CD)。通过对核磁谱图积分计算可以得到β-环糊精修饰透明质酸分子式为(C14H21NO11)98(C58H95N3O44)17,平均一条高分子链上修饰17个β-环糊精单元。Detection shows that the NMR characterization of the β-cyclodextrin-modified hyaluronic acid prepared by the present invention is as follows:1 H NMR (400MHz, D2 O, TMS, ppm): δ1.97 (s, 3H, H of methyl group of HA) , 2.84-3.93 (m, 16.24H, H of HA and C-3, C-5, C-6, C-2, C-4 of β-CD), 4.45-4.50 (m, 2H, H of HA), 5.04 -5.10 (m, 1.06H, H ofC-1ofβ-CD). The molecular formula of β-cyclodextrin-modified hyaluronic acid is (C14 H21 NO11 )98 (C58 H95 N3 O44 )17 through the integral calculation of the NMR spectrum, with an average of 17 modifications on one polymer chain. β-cyclodextrin unit.
(2)金刚烷多胺化合物的制备(2) Preparation of adamantane polyamine compound
1)将265mg(1.3mmol)二叠氮甲基苯酚化合物(化合物1)、247mg(0.962mmol)金刚烷溴甲基酮和540mg(3.9mmol)无水碳酸钾在50mL丙酮中混合，加热至56℃回流搅拌反应12h，然后过滤，将滤液旋干，加入50mL三氯甲烷和50mL水进行萃取，有机相用无水氯化镁干燥，减压蒸馏除去溶剂得到粗品，然后以石油醚和乙酸乙酯混合液为展开剂，该混合液中石油醚和乙酸乙酯的体积比为8:1，用200-300目硅胶柱进行分离即可制得二叠氮甲基苯基金刚烷化合物(化合物2)的纯品为无色油状物；1) Mix 265mg (1.3mmol) diazide methylphenol compound (compound 1), 247mg (0.962mmol) adamantyl bromomethyl ketone and 540mg (3.9mmol) anhydrous potassium carbonate in 50mL acetone, heat to 56 ℃ reflux and stir for 12 hours, then filter, spin the filtrate to dryness, add 50mL chloroform and 50mL water for extraction, dry the organic phase with anhydrous magnesium chloride, distill off the solvent under reduced pressure to obtain the crude product, then mix it with petroleum ether and ethyl acetate liquid is a developing agent, and the volume ratio of sherwood oil and ethyl acetate in the mixed solution is 8:1, and the diazidemethylphenyladamantane compound (compound 2) can be obtained by separating with a 200-300 mesh silica gel column. The pure product is a colorless oil;
检测显示本发明制备的二叠氮甲基苯基金刚烷化合物(化合物2)的核磁表征如下：1H NMR(400MHz,CDCl3,ppm)δ6.87(s,1H),6.79(s,2H),4.89(s,2H),4.33(s,4H),2.10(m,3H),1.94(m,6H),1.78(m,6H)。Detection shows that the NMR characterization of the diazidemethylphenyladamantane compound (compound 2) prepared by the present invention is as follows:1 H NMR (400MHz, CDCl3 , ppm) δ6.87(s, 1H), 6.79(s, 2H ), 4.89(s,2H), 4.33(s,4H), 2.10(m,3H), 1.94(m,6H), 1.78(m,6H).
2)将738mg(1.94mmol)二叠氮甲基苯基金刚烷化合物(化合物2)和1563mg(5.96mmol)三苯基膦溶于20mL无水四氢呋喃中，在室温条件下搅拌12小时，然后减压蒸馏除去溶剂，将所得固体溶于乙酸乙酯中，加入0.5mL、浓度为36.5wt％的盐酸后得到白色沉淀，收集沉淀并用乙酸乙酯洗涤，得到二胺甲基苯基金刚烷化合物(化合物3)的纯品；2) 738mg (1.94mmol) diazidemethylphenyladamantane compound (compound 2) and 1563mg (5.96mmol) triphenylphosphine were dissolved in 20mL of anhydrous THF, stirred at room temperature for 12 hours, then reduced Pressure distillation removes solvent, gained solid is dissolved in the ethyl acetate, after adding 0.5mL, concentration is the hydrochloric acid of 36.5wt% to obtain white precipitate, collect precipitate and wash with ethyl acetate, obtain diaminomethylphenyladamantane compound ( Compound 3) pure product;
检测显示本发明制备的二胺甲基苯基金刚烷化合物(化合物3)的核磁表征如下：1H NMR(400MHz,D2O,ppm)δ7.00(s,1H),6.92(s,2H),5.09(s,2H),4.05(s,4H),1.94(m,3H),1.80(m,6H),1.61(m,6H)。Detection shows that the NMR characterization of the diaminomethylphenyladamantane compound (compound 3) prepared by the present invention is as follows:1 H NMR (400MHz, D2 O, ppm) δ7.00(s, 1H), 6.92(s, 2H ), 5.09(s,2H), 4.05(s,4H), 1.94(m,3H), 1.80(m,6H), 1.61(m,6H).
3)将120mg(0.3mmol)二胺甲基苯基金刚烷化合物(化合物3)、400mg(0.69mmol)氯甲酸苄酯保护的五氟苯酚多胺化合物(化合物4)和0.2mL三乙胺溶于20mL二氯甲烷中，室温下搅拌反应20小时，减压蒸馏除去溶剂，加入50mL二氯甲烷和50mL水进行萃取，有机相用无水硫酸镁干燥，然后减压蒸馏除去溶剂得到粗品，以乙酸乙酯和石油醚混合液为展开剂，该混合液中石油醚和乙酸乙酯的体积比依次为1:2变至4:1变至6:1最后为纯乙酸乙酯，用200-300目硅胶柱进行分离，制得氯甲酸苄酯保护的金刚烷多胺化合物(化合物5)的纯品为白色固体；3) 120mg (0.3mmol) diaminomethylphenyladamantane compound (compound 3), 400mg (0.69mmol) benzyl chloroformate protected pentafluorophenol polyamine compound (compound 4) and 0.2mL triethylamine were dissolved In 20mL of dichloromethane, stirred and reacted at room temperature for 20 hours, distilled off the solvent under reduced pressure, added 50mL of dichloromethane and 50mL of water for extraction, dried the organic phase with anhydrous magnesium sulfate, and then distilled off the solvent under reduced pressure to obtain a crude product. The mixed solution of ethyl acetate and petroleum ether is used as the developer, and the volume ratio of petroleum ether and ethyl acetate in the mixed solution is sequentially changed from 1:2 to 4:1 to 6:1 and finally pure ethyl acetate, using 200-300 Mesh silica gel column is separated, and the pure product of the adamantane polyamine compound (compound 5) that makes benzyl chloroformate protection is white solid;
检测显示本发明制备的氯甲酸苄酯保护的金刚烷多胺化合物(化合物5)的核磁表征如下：1H NMR(400MHz,CDCl3,ppm)δ7.32(m,20H),6.62(m,3H),5.11(s,4H),5.05(s,4H),4.80(s,2H),4.31(m,4H),3.90(s,4H).3.33(m,4H),3.10(m,4H),2.05(m,3H),1.88(m,6H),1.72(m,6H),1.52-1.42(m,8H)。Detection shows that the NMR characterization of the adamantane polyamine compound (compound 5) prepared by the present invention protected by benzyl chloroformate is as follows:1 H NMR (400MHz, CDCl3 , ppm) δ7.32 (m, 20H), 6.62 (m, 3H),5.11(s,4H),5.05(s,4H),4.80(s,2H),4.31(m,4H),3.90(s,4H).3.33(m,4H),3.10(m,4H ), 2.05(m,3H), 1.88(m,6H), 1.72(m,6H), 1.52-1.42(m,8H).
4)将211.5mg(0.2mmol)氯甲酸苄酯保护的金刚烷多胺化合物(化合物5)溶于5mL氢溴酸与乙酸的混合溶液中，氢溴酸在与乙酸的混合溶液中的浓度为33wt％，室温搅拌反应10小时，加入过量的乙醚产生白色沉淀，离心并用乙醚进行洗涤即可制得金刚烷多胺化合物的纯品为白色固体；4) 211.5mg (0.2mmol) benzyl chloroformate protected adamantane polyamine compound (compound 5) is dissolved in the mixed solution of 5mL hydrobromic acid and acetic acid, the concentration of hydrobromic acid in the mixed solution with acetic acid is 33wt%, stirred and reacted at room temperature for 10 hours, added excessive ether to produce a white precipitate, centrifuged and washed with ether to obtain the pure product of adamantane polyamine compound as a white solid;
检测显示本发明制备的金刚烷多胺化合物的核磁表征如下：1H NMR(400MHz,CDCl3,ppm)δ6.66(s,1H),6.51(s,2H),4.94(s,2H),4.15(s,4H),3.69(s,4H),2.88(m,4H),2.78(m,4H).1.82(m,3H),1.68(m,6H),1.54(m,14H)。Detection shows that the nuclear magnetic characterization of the adamantane polyamine compound prepared by the present invention is as follows:1 H NMR (400MHz, CDCl3 , ppm) δ6.66 (s, 1H), 6.51 (s, 2H), 4.94 (s, 2H), 4.15 (s, 4H), 3.69 (s, 4H), 2.88 (m, 4H), 2.78 (m, 4H). 1.82 (m, 3H), 1.68 (m, 6H), 1.54 (m, 14H).
(3)靶向传递小干扰RNA的超分子组装体的制备(3) Preparation of supramolecular assemblies for targeted delivery of small interfering RNA
将1.20mgβ-环糊精修饰透明质酸、0.29mg金刚烷多胺化合物和0.64mg葫芦脲[6]混合并溶于80μL水中，进行超声溶解，然后加入80μL浓度为20μM小干扰RNA的水溶液，用200μL移液枪反复吹打20次，制得靶向传递小干扰RNA的四元超分子组装体。Mix 1.20 mg of β-cyclodextrin-modified hyaluronic acid, 0.29 mg of adamantane polyamine compound, and 0.64 mg of cucurbituril [6] and dissolve them in 80 μL of water, perform ultrasonic dissolution, and then add 80 μL of an aqueous solution with a concentration of 20 μM small interfering RNA, Use a 200 μL pipette gun to repeatedly pipette 20 times to prepare a quaternary supramolecular assembly for targeted delivery of small interfering RNA.
图1为金刚烷多胺化合物的合成路线图。Figure 1 is a synthetic route diagram of adamantane polyamine compounds.
图2为该四元超分子组装体的构筑路线示意图。Figure 2 is a schematic diagram of the construction route of the quaternary supramolecular assembly.
图3为该四元超分子组装体的透射电子显微镜图，通过透射电子显微镜表征可以得出该四元超分子组装体以环糊精-金刚烷、正电荷多胺-葫芦脲[6]之间强的非共价相互作用以及正电荷多胺-小干扰RNA间静电相互作用为基础形成的超分子纳米粒子，纳米粒子粒径大小为30-40nm。Figure 3 is a transmission electron microscope image of the quaternary supramolecular assembly. Through the characterization of the transmission electron microscope, it can be concluded that the quaternary supramolecular assembly is composed of cyclodextrin-adamantane, positively charged polyamine-cucurbituril [6]. Supramolecular nanoparticles formed on the basis of strong non-covalent interactions and electrostatic interactions between positively charged polyamines and small interfering RNAs. The size of the nanoparticles is 30-40nm.
图4为该四元超分子组装体与小干扰RNA键合的电泳图。相比单纯的小干扰RNA(第1列)和小干扰RNA+β-环糊精修饰透明质酸+金刚烷多胺化合物(第2列)复合物，葫芦脲[6]的加入能够有效增强超分子组装体对小干扰RNA的键合能力(3-5列)，最后化学计量比下的β-环糊精修饰透明质酸+金刚烷多胺化合物+葫芦脲[6](β-环糊精修饰透明质酸、金刚烷多胺化合物和葫芦脲[6]的摩尔比为1:17:34)对小干扰RNA的键合能力最强(第6列)。Fig. 4 is an electrophoresis diagram of the binding of the quaternary supramolecular assembly to the small interfering RNA. Compared with simple small interfering RNA (column 1) and complexes of small interfering RNA + β-cyclodextrin modified hyaluronic acid + adamantane polyamine compound (column 2), the addition of cucurbituril [6] can effectively enhance The binding ability of supramolecular assembly to small interfering RNA (columns 3-5), the final stoichiometric ratio of β-cyclodextrin modified hyaluronic acid + adamantane polyamine compound + cucurbituril [6] (β-cyclo The molar ratio of dextrin-modified hyaluronic acid, adamantane polyamine compound and cucurbituril [6] was 1:17:34) had the strongest binding ability to small interfering RNA (column 6).
本发明的具体应用效果如下：Concrete application effect of the present invention is as follows:
将PC-3细胞(人类前列腺癌细胞)铺在含有10％胎牛血清的RPMI-1640培养基的96孔板中培养24小时，分别加入160μL，80μL，40μL，20μL的金刚烷多胺化合物(a)，金刚烷多胺化合物+葫芦脲[6](b)，金刚烷多胺化合物+β-环糊精修饰透明质酸(c)，以及葫芦脲[6]+金刚烷多胺化合物+β-环糊精修饰透明质酸(d)，连续培养24小时，用MTT法测量各个实验条件下的细胞生存率。结果如图5所示，在24小时范围内，各个浓度下的金刚烷多胺化合物由于其具有带正电荷的多胺链而展现出了一定的细胞毒性，而加入β-环糊精修饰透明质酸后，由于透明质酸的靶向作用，使得β-环糊精修饰透明质酸-金刚烷多胺化合物组装体大量被内吞进细胞内，使得金刚烷多胺化合物的细胞毒性得到增强。而葫芦脲[6]的加入使其键合在正电荷多胺链上，能够大大降低组装体的细胞毒性,细胞生存率普遍在90％以上。由此可见，我们构筑的四元超分子组装体在各个浓度下展现出了很低的细胞毒性，安全性大幅提升，有利于后面基因传递效果的评价。另一方面，如图6所示，将PC-3细胞铺在含有10％胎牛血清的RPMI-1640培养基的6孔板中培养24小时，预先用Lipofactamine 2000和EGFP-pDNA处理细胞使得细胞表达出绿色荧光蛋白，然后分别加入Lipofacyamine 2000+EGFP-小干扰RNA(d-f)和负载EGFP-小干扰RNA的四元超分子组装体(g-i)，作用6小时后给细胞换成新鲜培养基，再培养24小时，用荧光显微镜观察绿色荧光蛋白基因的沉默效果。如图6a-6c所示，PC-3细胞在转入EGFP-pDNA后有效表达出了绿色荧光蛋白，继而展现出了明亮的绿色荧光；商品化试剂Lipofacyamine 2000键合EGFP-小干扰RNA展现出了良好的对绿色荧光蛋白的沉默效率，而我们制备的负载EGFP-小干扰RNA的四元超分子组装体能够有效将EGFP-小干扰RNA转染进入PC-3细胞内，使得荧光细胞数量和细胞荧光强度大大降低，显示出了比商品化试剂更好的对绿色荧光蛋白的基因沉默效果，具有很好的基因传递应用前景。PC-3 cells (human prostate cancer cells) were plated in 96-well plates containing 10% fetal bovine serum RPMI-1640 medium for 24 hours, and 160 μL, 80 μL, 40 μL, and 20 μL of adamantane polyamine compounds ( a), adamantane polyamine compound + cucurbituril [6] (b), adamantane polyamine compound + β-cyclodextrin modified hyaluronic acid (c), and cucurbituril [6] + adamantane polyamine compound + β-cyclodextrin modified hyaluronic acid (d), cultured continuously for 24 hours, and measured cell viability under various experimental conditions by MTT method. The results are shown in Figure 5. Within 24 hours, the adamantane polyamine compounds at various concentrations exhibited certain cytotoxicity due to their positively charged polyamine chains, while adding β-cyclodextrin to modify the transparent After hyaluronic acid, due to the targeting effect of hyaluronic acid, a large number of β-cyclodextrin-modified hyaluronic acid-adamantane polyamine compound assemblies are endocytosed into cells, which enhances the cytotoxicity of adamantane polyamine compounds . The addition of cucurbituril [6] makes it bonded to the positively charged polyamine chain, which can greatly reduce the cytotoxicity of the assembly, and the cell survival rate is generally above 90%. It can be seen that the quaternary supramolecular assembly we constructed exhibited very low cytotoxicity at various concentrations, and the safety was greatly improved, which is conducive to the evaluation of the subsequent gene delivery effect. On the other hand, as shown in Figure 6, PC-3 cells were cultured in 6-well plates containing RPMI-1640 medium containing 10% fetal bovine serum for 24 hours, and the cells were pre-treated with Lipofactamine 2000 and EGFP-pDNA to make the cells Express green fluorescent protein, then add Lipofacyamine 2000+EGFP-small interfering RNA (d-f) and quaternary supramolecular assembly (g-i) loaded with EGFP-small interfering RNA respectively, and replace the cells with fresh medium after acting for 6 hours. After culturing for another 24 hours, the silencing effect of the green fluorescent protein gene was observed with a fluorescence microscope. As shown in Figures 6a-6c, PC-3 cells effectively expressed green fluorescent protein after transfection with EGFP-pDNA, and then showed bright green fluorescence; the commercial reagent Lipofacyamine 2000 bonded with EGFP-small interfering RNA showed It has a good silencing efficiency for green fluorescent protein, and the quaternary supramolecular assembly loaded with EGFP-small interfering RNA prepared by us can effectively transfect EGFP-small interfering RNA into PC-3 cells, so that the number of fluorescent cells and The fluorescence intensity of cells is greatly reduced, showing a better gene silencing effect on green fluorescent protein than commercial reagents, and has a good prospect for gene delivery applications.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510200845.XACN104840975B (en) | 2015-04-24 | 2015-04-24 | A kind of super-molecule assembling body and preparation method of targeted delivery siRNA |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510200845.XACN104840975B (en) | 2015-04-24 | 2015-04-24 | A kind of super-molecule assembling body and preparation method of targeted delivery siRNA |
| Publication Number | Publication Date |
|---|---|
| CN104840975A CN104840975A (en) | 2015-08-19 |
| CN104840975Btrue CN104840975B (en) | 2018-01-23 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510200845.XAExpired - Fee RelatedCN104840975B (en) | 2015-04-24 | 2015-04-24 | A kind of super-molecule assembling body and preparation method of targeted delivery siRNA |
| Country | Link |
|---|---|
| CN (1) | CN104840975B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105999289B (en)* | 2016-04-01 | 2018-11-20 | 南开大学 | A kind of super amphiphilic nano particle solution of binary and its preparation method and application |
| CN108659229B (en)* | 2017-04-01 | 2021-07-20 | 香港大学 | A kind of active supramolecular polymer and preparation method thereof |
| CN109082457A (en)* | 2018-05-10 | 2018-12-25 | 中国科学院武汉物理与数学研究所 | A kind of hyperpolarization 129Xe switching mode magnetic resonance molecular probe identifying diamine oxidase |
| CN109091678B (en)* | 2018-08-21 | 2022-01-28 | 南开大学 | Preparation method and application of double-regulation supermolecule assembly for inhibiting tumor invasion and diffusion |
| CN110152017A (en)* | 2019-04-28 | 2019-08-23 | 内蒙古农业大学 | A nanographene-based multifunctional supramolecular assembly and its preparation method and application |
| CN110478322B (en)* | 2019-09-17 | 2021-07-27 | 国家纳米科学中心 | A kind of nucleic acid drug complex and its preparation method and application |
| KR102243448B1 (en)* | 2019-10-30 | 2021-04-22 | 한국과학기술연구원 | Positively charged nanoparticles, uses and preparation methods thereof |
| CN111647165B (en)* | 2020-06-28 | 2021-09-28 | 天津科技大学 | Preparation method and application of dendritic supramolecular polymer based on cyclodextrin |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI321054B (en)* | 2000-12-19 | 2010-03-01 | California Inst Of Techn | Compositions containing inclusion complexes |
| CN102698286B (en)* | 2012-07-02 | 2013-10-09 | 南开大学 | A supramolecular assembly and its preparation for targeted delivery of adamantane cisplatin anticancer prodrug |
| KR101605528B1 (en)* | 2012-12-13 | 2016-03-23 | 포항공과대학교 산학협력단 | Novel hydrogel based on hyaluronic acid and use thereof |
| CN103550786B (en)* | 2013-11-08 | 2015-02-11 | 南开大学 | Supermolecular nano-assembly for gene therapy and preparation method thereof |
| CN103920160B (en)* | 2014-04-25 | 2016-02-03 | 南开大学 | Cyclodextrin is Graphene/hyaluronic acid assembly of medium and preparation method thereof |
| Publication number | Publication date |
|---|---|
| CN104840975A (en) | 2015-08-19 |
| Publication | Publication Date | Title |
|---|---|---|
| CN104840975B (en) | A kind of super-molecule assembling body and preparation method of targeted delivery siRNA | |
| Tang et al. | Cell-selective messenger RNA delivery and CRISPR/Cas9 genome editing by modulating the interface of phenylboronic acid-derived lipid nanoparticles and cellular surface sialic acid | |
| Ortega et al. | Exosomes as drug delivery systems: endogenous nanovehicles for treatment of systemic lupus erythematosus | |
| Dong et al. | Multifunctional poly (l-lactide)–polyethylene glycol-grafted graphene quantum dots for intracellular microRNA imaging and combined specific-gene-targeting agents delivery for improved therapeutics | |
| Yi et al. | Nanoscale zeolitic imidazolate framework-8 for ratiometric fluorescence imaging of microRNA in living cells | |
| JP7683017B2 (en) | Ionizable lipid molecules, their preparation and application in the preparation of lipid nanoparticles | |
| CN102698286B (en) | A supramolecular assembly and its preparation for targeted delivery of adamantane cisplatin anticancer prodrug | |
| CN110746599B (en) | UV (ultraviolet) light-responsive hyperbranched poly-beta-amino ester with high-efficiency gene delivery capacity as well as preparation method and application thereof | |
| CN104352440A (en) | Cationic liposome nucleic acid medicinal preparation as well as preparation method and application thereof | |
| CN103263673B (en) | Polysaccharide-gold-nanoparticle supermolecule assembled body as well as preparation method and application thereof | |
| CN114904003B (en) | Use of ionizable cationic lipid analog materials as nucleic acid drug delivery vehicles or transfection reagents | |
| CN107936058B (en) | Docetaxel derivative and preparation method and application thereof | |
| CN102258788B (en) | Targeted transmission assembly of adriamycin anticancer medicine and preparation method thereof | |
| Yang et al. | Novel pH-responsive biodegradable organosilica nanoparticles as drug delivery system for cancer therapy | |
| CN103665222A (en) | Cation functional polymers with natural amino acid as side group, preparation method and application of polymer | |
| Shen et al. | Polyphosphoester-based cationic nanoparticles serendipitously release integral biologically-active components to serve as novel degradable inducible nitric oxide synthase inhibitors | |
| CN101870719B (en) | Organic functional compound having disulfide chemical bond and steroids skeleton, preparation method thereof and use thereof | |
| CN116803995B (en) | A class of aminoamide lipid compounds and preparation methods and applications thereof | |
| Fang et al. | Novel mitochondrial targeting charge-reversal polysaccharide hybrid shell/core nanoparticles for prolonged systemic circulation and antitumor drug delivery | |
| Korzuch et al. | Towards water-soluble [60] fullerenes for the delivery of siRNA in a prostate cancer model | |
| CN111269161B (en) | Honokiol and sulforaphane conjugation and preparation method and application thereof | |
| CN103159819A (en) | Synthesis of cholesterol derivative and application thereof in gene transfection | |
| CN103570942A (en) | Polyethyleneimine function cation polymer derived from natural cholesterol, synthesis method and uses thereof | |
| CN108635590B (en) | PH-responsive polysaccharide-bortezomib nanosphere and preparation method and application thereof | |
| Saneyoshi et al. | Design, synthesis, and cellular uptake of oligonucleotides bearing glutathione-labile protecting groups |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate 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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee | Granted publication date:20180123 Termination date:20190424 |