技术领域Technical field
本发明属于生物医药技术领域，具体而言属于一种新型可电离阳离子脂质化合物、制备方法及应用。The invention belongs to the field of biomedicine technology, specifically a new type of ionizable cationic lipid compound, preparation method and application.
背景技术Background technique
脂质体作为脂质纳米颗粒(LNP)的早期版本出现，成为一种多功能的纳米载体平台，能够运输疏水和亲水药物，包括小分子化学药物、抗体药物和核酸药物。脂质体代表了纳米药物递送系统从概念到临床应用的首次成功转化。与脂质体相比，LNP表现出更复杂的结构和更强的物理稳定性，使基因编辑和蛋白质替代成为可能。Liposomes emerged as an early version of lipid nanoparticles (LNPs) and became a versatile nanocarrier platform capable of transporting hydrophobic and hydrophilic drugs, including small molecule chemical drugs, antibody drugs, and nucleic acid drugs. Liposomes represent the first successful translation of a nanodrug delivery system from concept to clinical application. Compared with liposomes, LNPs exhibit a more complex structure and stronger physical stability, making gene editing and protein replacement possible.
目前FDA批准的所有LNP制剂均基于四种脂质：可电离阳离子脂质、磷脂、胆固醇和PEG脂质组成，每种成分发挥不同作用。这些成分促进了LNP的形成以及核酸的高效包封，同时也提高了LNP的稳定性，增加了核酸的细胞摄取，促进了核酸药物的溶酶体逃逸。在众多的因素之中，可电离阳离子脂质的结构是影响LNP递送效率的直接因素。All LNP formulations currently approved by the FDA are based on four lipids: ionizable cationic lipids, phospholipids, cholesterol and PEG lipids, with each component playing a different role. These ingredients promote the formation of LNP and efficient encapsulation of nucleic acids, while also improving the stability of LNP, increasing the cellular uptake of nucleic acids, and promoting the lysosomal escape of nucleic acid drugs. Among many factors, the structure of ionizable cationic lipids is a direct factor affecting LNP delivery efficiency.
裸露的RNA是一种带负电荷的亲水性大分子，由于细胞膜的静电排斥，难以进入细胞，且易被体内RNA酶迅速降，需要保护性外壳才能进入细胞。因为细胞膜主要由脂质组成，利用脂质囊泡包封RNA可通过细胞膜并将RNA释放到细胞质中。因此，囊泡首先应该是一种带正电的脂质，能够结合带负电的RNA。然而，由永久性阳离子脂质组成的囊泡会与带负电的细胞膜发生静电作用而引起细胞毒性，且目前报道中所使用的LNP的阳离子脂质被细胞吞噬的仅有1-4％可成功实现溶酶体逃逸，且所使用的阳离子脂质所制备的LNP对siRNA转染能力较低，所以仍然需要对用于递送核酸药物的可电离阳离子脂质进行改进。Naked RNA is a negatively charged hydrophilic macromolecule. Due to the electrostatic repulsion of the cell membrane, it is difficult to enter the cell and is easily degraded by RNase in the body. It requires a protective shell to enter the cell. Because the cell membrane is mainly composed of lipids, using lipid vesicles to encapsulate RNA can pass through the cell membrane and release the RNA into the cytoplasm. Therefore, the vesicle should first be a positively charged lipid capable of binding negatively charged RNA. However, vesicles composed of permanent cationic lipids can cause electrostatic interaction with negatively charged cell membranes and cause cytotoxicity, and only 1-4% of the cationic lipids of LNP used in current reports can be successfully engulfed by cells. Lysosomal escape is achieved, and the LNPs prepared from the cationic lipids used have low siRNA transfection ability, so there is still a need to improve the ionizable cationic lipids used to deliver nucleic acid drugs.
有鉴于此，特提出本发明。In view of this, the present invention is proposed.
发明内容Contents of the invention
本发明的第一目的在于提供一种可电离阳离子脂质化合物，该种化合物可与其他脂质组分例如中性脂质、带电脂质、类固醇(包括例如，所有固醇类)和/或它们的类似物、和/或聚合物缀合的脂质联合使用，以形成用于递送治疗剂的脂质纳米颗粒。对载siRNA脂质纳米颗粒具有更高的转染率，更适合应用到核酸药物领域中。A first object of the present invention is to provide an ionizable cationic lipid compound that can be combined with other lipid components such as neutral lipids, charged lipids, steroids (including, for example, all sterols) and/or Their analogs, and/or polymer-conjugated lipids are used in combination to form lipid nanoparticles for delivery of therapeutic agents. Lipid nanoparticles loaded with siRNA have a higher transfection rate and are more suitable for application in the field of nucleic acid drugs.
本发明的第二目的在于提供上述型可电离阳离子脂质化合物的制备方法，该种方法可以以高收率得到上述可电离阳离子脂质化合物。The second object of the present invention is to provide a method for preparing the above-mentioned ionizable cationic lipid compound, which method can obtain the above-mentioned ionizable cationic lipid compound in high yield.
为实现本发明的上述目的，特采用以下技术方案：In order to achieve the above objects of the present invention, the following technical solutions are adopted:
一种阳离子脂质化合物，其特征在于，所述阳离子脂质化合物的化学结构A cationic lipid compound, characterized in that the chemical structure of the cationic lipid compound
通式为其中，The general formula is in,
R1’、R2’、R3’和R4均为烷基；R1 ', R2 ', R3 ' and R4 are all alkyl groups;
M，M1为酯基；M, M1 is ester group;
j为CH2的数量，为2,3,4其中的任意一种；j is the number of CH2 , which is any one of 2, 3, and 4;
n为CH2的数量，为2,3,4其中的任意一种。n is the number of CH2 , which is any one of 2, 3, and 4.
优选地，作为进一步具体的实施方式，Preferably, as a further specific implementation,
R1’为C8-10含醚键的烷基；R1 ' is a C8-10 alkyl group containing an ether bond;
R2’为C6-8的烷基；R2 ' is a C6-8 alkyl group;
R3’为C6-8的烷基；R3 ' is a C6-8 alkyl group;
R4为含羟基的烷基，为羟甲基，羟乙基，羟丙基中的任意一种。R4 is a hydroxyl-containing alkyl group, which is any one of hydroxymethyl, hydroxyethyl and hydroxypropyl.
本发明还提供了一种阳离子脂质化合物的制备方法，反应通式如下：The invention also provides a method for preparing a cationic lipid compound. The general reaction formula is as follows:
优选地，作为进一步具体的实施方式，A过程反应温度为25℃-30℃，反应时间为3.5h-4h。Preferably, as a further specific implementation, the reaction temperature of process A is 25°C-30°C, and the reaction time is 3.5h-4h.
优选地，作为进一步具体的实施方式，B过程反应温度为25℃-30℃，反应时间为24h-28h。Preferably, as a further specific implementation, the reaction temperature of process B is 25°C-30°C, and the reaction time is 24h-28h.
优选地，作为进一步具体的实施方式，C过程反应温度为25℃-30℃，反应时间为3h-5h。Preferably, as a further specific embodiment, the reaction temperature of process C is 25°C-30°C, and the reaction time is 3h-5h.
优选地，作为进一步具体的实施方式，D过程反应温度为60℃-65℃，反应时间为48h-50h。Preferably, as a further specific implementation, the reaction temperature of process D is 60°C-65°C, and the reaction time is 48h-50h.
优选地，作为进一步具体的实施方式，反应所述的烷基(C13-17)醇的羟基取代基的位置为所选烷基(C13-17)醇上碳原子的任意位。Preferably, as a further specific embodiment, the position of the hydroxyl substituent of the alkyl (C13-17 ) alcohol described in the reaction is any position of the carbon atom on the selected alkyl (C13-17 ) alcohol.
本发明所设计的新型可电离阳离子脂质化合物，通过测量LNP的pKa，安全性，体外转染效率和体外脏器分布。研究结果表明，设计有醚键加入的阳离子脂质(化合物I)中LNP的pKa值均在6.5～6.7之间，说明在疏水尾部加入醚键可电离脂质会提高LNP递送siRNA的效率。另外，细胞毒性和溶血实验显示，化合物I醚键的加入不会对LNPs产生任何额外的毒性，制备的LNP具有良好的生物安全性和生物相容性。体外转染效率结果显示，化合物lipoND系列基因沉默效率较好，转染效率和肝脏靶向性能也较好。The novel ionizable cationic lipid compound designed by the present invention is tested by measuring the pKa, safety, in vitro transfection efficiency and in vitro organ distribution of LNP. Research results show that the pKa values of LNPs in cationic lipids (Compound I) designed with ether bonds added are between 6.5 and 6.7, indicating that adding ether bond-ionizable lipids to the hydrophobic tail will improve the efficiency of LNP delivery of siRNA. In addition, cytotoxicity and hemolysis experiments show that the addition of the ether bond of Compound I will not cause any additional toxicity to LNPs, and the prepared LNPs have good biosafety and biocompatibility. In vitro transfection efficiency results show that the lipoND series of compounds have better gene silencing efficiency, as well as better transfection efficiency and liver targeting performance.
本发明还提供了合成上述化合物的制备方法，该方法，可以以高收率得到得到了目标化合物。The present invention also provides a preparation method for synthesizing the above compound. This method can obtain the target compound in high yield.
与现有技术相比，本发明的有益效果在于：Compared with the prior art, the beneficial effects of the present invention are:
(1)本发明提供了一种醚类脂质化合物、其组合物、制备方法及应用。可与其他脂质组分例如中性脂质、带电脂质、类固醇(包括例如，所有固醇类)和/或它们的类似物、和/或聚合物缀合的脂质联合使用，以形成用于递送治疗剂的脂质纳米颗粒。对载siRNA脂质纳米颗粒具有更高的转染率，提高了LNP递送siRNA的效率，更适合应用到核酸药物领域中。(1) The present invention provides an ether lipid compound, its composition, preparation method and application. Can be used in combination with other lipid components such as neutral lipids, charged lipids, steroids (including, for example, all sterols) and/or their analogs, and/or polymer-conjugated lipids to form Lipid nanoparticles for delivery of therapeutic agents. Lipid nanoparticles loaded with siRNA have a higher transfection rate, improve the efficiency of LNP delivery of siRNA, and are more suitable for application in the field of nucleic acid drugs.
(2)本发明所提供的新型阳离子脂质化合物，能够具有良好的生物安全性和生物相容性的同时安全无害，有较高的安全性。(2) The novel cationic lipid compound provided by the present invention can have good biological safety and biocompatibility while being safe and harmless, and has high safety.
附图说明Description of drawings
图1：化合物lipoND-1的H谱图；图2：化合物lipoND-2的H谱图；Figure 1: H spectrum of compound lipoND-1; Figure 2: H spectrum of compound lipoND-2;
图3：化合物lipoND-3的H谱图；图4：化合物lipoND-4的H谱图；Figure 3: H spectrum of compound lipoND-3; Figure 4: H spectrum of compound lipoND-4;
图5：化合物lipoND-5的H谱图；Figure 5: H spectrum of compound lipoND-5;
图6：化合物lipoND-1的pKa值；图7：化合物lipoND-2的pKa值；Figure 6: pKa value of compound lipoND-1; Figure 7: pKa value of compound lipoND-2;
图8：化合物lipoND-3的pKa值；图9：化合物lipoND-4的pKa值；Figure 8: pKa value of compound lipoND-3; Figure 9: pKa value of compound lipoND-4;
图10：化合物lipoND-5的pKa值；图11：化合物lipid5的pKa值；Figure 10: pKa value of compound lipoND-5; Figure 11: pKa value of compound lipid5;
图12：SM-102pKa值；Figure 12: SM-102pKa value;
图13a：化合物lipoND-1、lipoND-2、lipoND-3、lipoND-4、lipoND-5、lipid5和SM-102对HSC-T6细胞细胞毒性检测结果；Figure 13a: Cytotoxicity test results of compounds lipoND-1, lipoND-2, lipoND-3, lipoND-4, lipoND-5, lipid5 and SM-102 on HSC-T6 cells;
图13b：化合物lipoND-1、lipoND-2、lipoND-3、lipoND-4、lipoND-5、lipid5和SM-102对NIH-3T3细胞细胞毒性检测结果；Figure 13b: Cytotoxicity test results of compounds lipoND-1, lipoND-2, lipoND-3, lipoND-4, lipoND-5, lipid5 and SM-102 on NIH-3T3 cells;
图14：化合物lipoND-1、lipoND-2、lipoND-3、lipoND-4、lipoND-5、lipid5和SM-102对于NIH-3T3细胞的转染率；Figure 14: Transfection rate of compounds lipoND-1, lipoND-2, lipoND-3, lipoND-4, lipoND-5, lipid5 and SM-102 into NIH-3T3 cells;
图15：化合物lipoND-1、lipoND-2、lipoND-3、lipoND-4、lipoND-5、lipid5和SM-102对于脏的靶向性研究结果。Figure 15: Results of studies on the organ targeting of compounds lipoND-1, lipoND-2, lipoND-3, lipoND-4, lipoND-5, lipid5 and SM-102.
具体实施方式Detailed ways
下面将结合具体实施方式对本发明的技术方案进行清楚、完整地描述，但是本领域技术人员将会理解，下列所描述的实施例是本发明一部分实施例，而不是全部的实施例，仅用于说明本发明，而不应视为限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, those skilled in the art will understand that the embodiments described below are some, not all, of the embodiments of the present invention and are only used for illustrative of the invention and should not be construed as limiting the scope of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
实施例1：化合物lipoND-1的制备Example 1: Preparation of compound lipoND-1
步骤一：化合物1的制备Step 1: Preparation of Compound 1
将NaOH(14.67g，366.75mmol)与乙二醇(200mL)置于反应瓶中，氮气保护下加入66mL甲苯回流分水，NaOH溶解后继续加热至无水生成。降低反应温度至90℃，将1-溴己烷(40.1g，244.5mmol)溶于THF中，滴加入反应液中，30min内滴加完毕，90℃反应16小时，反应结束。反应液中加入乙酸乙酯，并用水洗涤，合并有机相，无水硫酸镁干燥，减压旋蒸，经柱层析纯化后，得化合物1，淡黄色液体23.76g。收率：66.5％。Place NaOH (14.67g, 366.75mmol) and ethylene glycol (200mL) in a reaction bottle, add 66mL of toluene under nitrogen protection and reflux to separate the water. After NaOH is dissolved, continue to heat until anhydrous is formed. Lower the reaction temperature to 90°C, dissolve 1-bromohexane (40.1g, 244.5mmol) in THF, add dropwise to the reaction solution, complete the dropwise addition within 30 minutes, react at 90°C for 16 hours, and the reaction is completed. Ethyl acetate was added to the reaction solution, and the mixture was washed with water. The organic phases were combined, dried over anhydrous magnesium sulfate, and evaporated under reduced pressure. After purification by column chromatography, compound 1 was obtained as a light yellow liquid, 23.76 g. Yield: 66.5%.
步骤二：化合物2的制备Step 2: Preparation of compound 2
将8-溴辛酸(24.18g，108.39mmol)与化合物1(13.2g，90.33mmol)加入反应瓶中，200mL二氯甲烷溶清后加入EDC(47.6g，248.32mmol)与DMAP(2.2g，18.066mmol)，30℃反应24小时，经TLC检测，反应完全，反应结束用二氯甲烷稀释反应液，并用饱和碳酸氢钠与饱和食盐水洗涤，分离有机层，无水硫酸镁干燥，减压旋蒸，柱层析纯化，得到化合物2，淡黄色液体28.6g。收率：90％。Add 8-bromooctanoic acid (24.18g, 108.39mmol) and compound 1 (13.2g, 90.33mmol) into the reaction bottle. Dissolve 200mL of methylene chloride and add EDC (47.6g, 248.32mmol) and DMAP (2.2g, 18.066 mmol) at 30°C for 24 hours. The TLC test showed that the reaction was complete. At the end of the reaction, the reaction solution was diluted with methylene chloride and washed with saturated sodium bicarbonate and saturated brine. The organic layer was separated, dried over anhydrous magnesium sulfate, and vortexed under reduced pressure. Evaporate and purify by column chromatography to obtain 28.6 g of compound 2 as a pale yellow liquid. Yield: 90%.
步骤三：化合物3的制备Step 3: Preparation of compound 3
将镁条(1.5g，61.3mmol)加入反应瓶中，加入200mL无水THF，加入溴辛烷(13g，67.43mmol)，2粒碘粒，加热70℃反应，至镁条大部分分解，缓慢滴加壬醛(8.7g，61.3mmol)，30min滴加完毕，滴加完毕后继续加热4小时，反应结束。稀盐酸调节pH至酸性，使用乙酸乙酯萃取，用水洗涤有机相，收集有机相，减压旋蒸，柱层析纯化，得化合物3，白色固体13g。收率：83.3％Add magnesium strip (1.5g, 61.3mmol) into the reaction bottle, add 200mL anhydrous THF, add bromooctane (13g, 67.43mmol), 2 iodine grains, heat to 70°C for reaction, until most of the magnesium strip decomposes, slowly Nonanal (8.7g, 61.3mmol) was added dropwise and the addition was completed in 30 minutes. After the addition was completed, heating was continued for 4 hours to complete the reaction. Adjust the pH to acidic with dilute hydrochloric acid, extract with ethyl acetate, wash the organic phase with water, collect the organic phase, rotary evaporate under reduced pressure, and purify by column chromatography to obtain compound 3, 13 g of a white solid. Yield: 83.3%
步骤四：Boc氨基辛酸的制备Step 4: Preparation of Boc aminocaprylic acid
将氨基辛酸(25g，157.04mmol)加入反应瓶中，加入250ml水，搅拌溶清后，加入500ml丙酮。加入Boc酸酐(37.3g，172.74mmol)，三乙胺(31.78g，314.08mmol)，30℃反应4小时。经TLC检测，反应结束，经后处理，得Boc氨基辛酸40.52g，室温下为透明粘稠液体，低温时凝固。收率：99.5％。Add aminocaprylic acid (25g, 157.04mmol) into the reaction bottle, add 250ml of water, stir and dissolve, then add 500ml of acetone. Add Boc acid anhydride (37.3g, 172.74mmol) and triethylamine (31.78g, 314.08mmol), and react at 30°C for 4 hours. After TLC detection, the reaction was completed. After post-processing, 40.52g of Boc aminooctanoic acid was obtained. It was a transparent viscous liquid at room temperature and solidified at low temperature. Yield: 99.5%.
步骤五：Boc氨基辛酸9-十七酯的制备Step 5: Preparation of Boc aminooctanoic acid 9-heptadecanate
将9-十七醇(12.5g，48.8mmol)加入反应瓶中，加入Boc氨基辛酸(15.2g，58.6mmol)，加入210ml二氯甲烷，搅拌溶清后加入35ml DIPEA，DMAP(1.19g，9.76mmol)，EDC(23.4g，122mmol)，30℃搅拌反应28小时，TLC监测反应进程，反应完全后，经后处理，柱层析纯化。得Boc氨基辛酸9-十七酯21g，常温下为淡黄色固体。收率：86.5％。Add 9-heptadecanol (12.5g, 48.8mmol) into the reaction bottle, add Boc aminooctanoic acid (15.2g, 58.6mmol), add 210ml dichloromethane, stir and dissolve, then add 35ml DIPEA, DMAP (1.19g, 9.76 mmol), EDC (23.4g, 122mmol), stir and react at 30°C for 28 hours, monitor the reaction progress with TLC, and after the reaction is complete, perform post-treatment and purify by column chromatography. 21g of Boc aminooctanoic acid 9-heptadecanate was obtained, which was a light yellow solid at room temperature. Yield: 86.5%.
步骤六：氨基辛酸9-十七酯的制备Step 6: Preparation of 9-heptadecanyl aminooctanoate
将Boc氨基辛酸9-十七酯19.7g加入反应瓶中，加入60mL二氯甲烷，搅拌溶清后加入三氟乙酸40mL，在30℃条件下反应5小时，TLC监测反应结束，经后处理，得到黄色油状物15g，收率：95.2％。Add 19.7g of Boc 9-heptadecayl aminooctanoate into the reaction bottle, add 60mL of methylene chloride, stir and dissolve, add 40mL of trifluoroacetic acid, and react at 30°C for 5 hours. TLC monitors the end of the reaction. After post-processing, 15 g of yellow oil was obtained, yield: 95.2%.
步骤七：化合物10的制备Step 7: Preparation of compound 10
将氨基辛酸9-十七酯(6g，15.08mmol)加入反应瓶中，在反应瓶中加入化合物2(3.18g，9.05mmol)，加入DIPEA(2.73g，21.11mmol)，乙醇6mL，在氮气保护的条件下，65℃反应50小时，经TLC检测，到达反应终点，经后处理，柱层析纯化得到化合物10，4g，淡黄色液体。收率：64.7％。Add 9-heptadecanyl aminooctanoate (6g, 15.08mmol) to the reaction flask, add compound 2 (3.18g, 9.05mmol) to the reaction flask, add DIPEA (2.73g, 21.11mmol), and 6 mL of ethanol, under nitrogen protection Under the conditions, the reaction was carried out at 65°C for 50 hours. After TLC detection, the reaction end point was reached. After post-treatment and column chromatography purification, compound 10, 4g, was obtained as a light yellow liquid. Yield: 64.7%.
步骤八：化合物lipoND-1的制备Step 8: Preparation of compound lipoND-1
将化合物10(4g，5.81mmol)加入反应瓶中，加入2-溴乙醇(1.08g，8.72mmol)，加入DIPEA(1.12g，8.72mmol)，在氮气保护的条件下62℃反应24小时，经TLC检测，到达反应终点，反应结束，向反应液中加入乙酸乙酯，饱和碳酸氢钠溶液洗涤，饱和食盐水洗涤，无水硫酸镁干燥，减压旋蒸，柱层析纯化得到化合物lipoND-1，3.08g淡黄色液体。收率：72.2％。Add compound 10 (4g, 5.81mmol) into the reaction bottle, add 2-bromoethanol (1.08g, 8.72mmol), add DIPEA (1.12g, 8.72mmol), and react at 62°C for 24 hours under nitrogen protection. After TLC detection, the reaction endpoint was reached. The reaction was completed. Add ethyl acetate to the reaction solution, wash with saturated sodium bicarbonate solution, wash with saturated brine, dry with anhydrous magnesium sulfate, rotary evaporate under reduced pressure, and purify by column chromatography to obtain the compound lipoND- 1. 3.08g light yellow liquid. Yield: 72.2%.
实施例2：化合物lipoND-2的制备Example 2: Preparation of compound lipoND-2
步骤一至步骤三与实施例1相同Steps 1 to 3 are the same as in Example 1
步骤四：Boc氨基辛酸的制备Step 4: Preparation of Boc aminocaprylic acid
将氨基辛酸(25g，157.04mmol)加入反应瓶中，加入250ml水，搅拌溶清后，加入500ml丙酮。加入Boc酸酐(37.3g，172.74mmol)，三乙胺(31.78g，314.08mmol)，25℃反应3.5小时。经TLC检测，反应结束，经后处理，得Boc氨基辛酸40.52g，室温下为透明粘稠液体，低温时凝固。收率：99.5％。Add aminocaprylic acid (25g, 157.04mmol) into the reaction bottle, add 250ml of water, stir and dissolve, then add 500ml of acetone. Add Boc acid anhydride (37.3g, 172.74mmol) and triethylamine (31.78g, 314.08mmol), and react at 25°C for 3.5 hours. After TLC detection, the reaction was completed. After post-processing, 40.52g of Boc aminooctanoic acid was obtained. It was a transparent viscous liquid at room temperature and solidified at low temperature. Yield: 99.5%.
步骤五：Boc氨基辛酸9-十七酯的制备Step 5: Preparation of Boc aminooctanoic acid 9-heptadecanate
将9-十七醇(12.5g，48.8mmol)加入反应瓶中，加入Boc氨基辛酸(15.2g，58.6mmol)，加入210ml二氯甲烷，搅拌溶清后加入35ml DIPEA，DMAP(1.19g，9.76mmol)，EDC(23.4g，122mmol)，25℃搅拌反应24小时，TLC监测反应进程，反应完全后，经后处理，柱层析纯化。得Boc氨基辛酸9-十七酯21g，常温下为淡黄色固体。收率：86.5％。Add 9-heptadecanol (12.5g, 48.8mmol) into the reaction bottle, add Boc aminooctanoic acid (15.2g, 58.6mmol), add 210ml dichloromethane, stir and dissolve, then add 35ml DIPEA, DMAP (1.19g, 9.76 mmol), EDC (23.4g, 122mmol), stir and react at 25°C for 24 hours, monitor the reaction progress with TLC, and after the reaction is complete, perform post-treatment and purify by column chromatography. 21g of Boc aminooctanoic acid 9-heptadecanate was obtained, which was a light yellow solid at room temperature. Yield: 86.5%.
步骤六：氨基辛酸9-十七酯的制备Step 6: Preparation of 9-heptadecanyl aminooctanoate
将Boc氨基辛酸9-十七酯19.7g加入反应瓶中，加入60mL二氯甲烷，搅拌溶清后加入三氟乙酸40mL，在25℃条件下反应3小时，TLC监测反应结束，经后处理，得到黄色油状物15g，收率：95.2％。Add 19.7g of Boc 9-heptadecayl aminooctanoate into the reaction bottle, add 60mL of methylene chloride, stir and dissolve, add 40mL of trifluoroacetic acid, and react at 25°C for 3 hours. TLC monitors the reaction to completion. After post-processing, 15 g of yellow oil was obtained, yield: 95.2%.
步骤七：化合物8的合成Step 7: Synthesis of Compound 8
将氨基辛酸9-十七酯(5g，12.57mmol)加入反应瓶中，在反应瓶中加入化合物M1(2.65g，7.54mmol)，加入DIPEA(2.23g，17.56mmol)，乙醇5mL，在氮气保护的条件下，60℃反应48小时，经TLC检测，到达反应终点，经后处理，柱层析纯化得到化合物8，收率：71.0％。Add 9-heptadecanyl aminooctanoate (5g, 12.57mmol) into the reaction flask, add compound M1 (2.65g, 7.54mmol) into the reaction flask, add DIPEA (2.23g, 17.56mmol), and 5 mL of ethanol, under nitrogen protection Under the conditions of 60°C, the reaction was carried out for 48 hours. After TLC detection, the reaction end point was reached. After post-treatment and column chromatography purification, compound 8 was obtained with a yield of 71.0%.
步骤八：化合物lipoND-2的合成Step 8: Synthesis of compound lipoND-2
将化合物8加入反应瓶中，加入2-溴乙醇，加入DIPEA，在氮气保护的条件下62℃反应24小时，经TLC检测，到达反应终点，反应结束，向反应液中加入乙酸乙酯，饱和碳酸氢钠溶液洗涤，饱和食盐水洗涤，无水硫酸镁干燥，减压旋蒸，柱层析纯化得到化合物lipoND-2，收率：72.2％。Add compound 8 to the reaction bottle, add 2-bromoethanol, add DIPEA, react at 62°C for 24 hours under nitrogen protection, and detect by TLC that the reaction end point is reached. The reaction is completed. Add ethyl acetate to the reaction solution and make it saturated. Washed with sodium bicarbonate solution, washed with saturated brine, dried over anhydrous magnesium sulfate, evaporated under reduced pressure, and purified by column chromatography to obtain compound lipoND-2, yield: 72.2%.
实施例3：化合物lipoND-3的制备Example 3: Preparation of compound lipoND-3
步骤一至步骤三与实施例1相同Steps 1 to 3 are the same as in Example 1
步骤四：Boc氨基己酸的制备Step 4: Preparation of Boc aminocaproic acid
将氨基己酸((10.8g，95.95mmol)加入反应瓶中，加入100ml水，搅拌溶清后，加入200ml丙酮。加入Boc酸酐(23.03g，105.55mmol)，三乙胺(19.42g，191.9mmol)，30℃反应4小时。经TLC检测，反应结束，将反应液减压旋蒸，除去丙酮，剩下的水相用稀盐酸调pH至2～3，乙酸乙酯萃取×4，收集有机相，饱和食盐水洗×2，无水硫酸镁干燥，减压旋蒸，得Boc氨基己酸22g，室温下为透明粘稠液体，低温时凝固。收率：99.7％。Add aminocaproic acid ((10.8g, 95.95mmol) into the reaction bottle, add 100ml of water, stir and dissolve, add 200ml of acetone. Add Boc anhydride (23.03g, 105.55mmol), triethylamine (19.42g, 191.9mmol) ), react at 30°C for 4 hours. After TLC detection, the reaction is completed. The reaction solution is evaporated under reduced pressure to remove acetone. Use dilute hydrochloric acid to adjust the pH of the remaining aqueous phase to 2-3. Extract with ethyl acetate × 4. Collect the organic matter. The phase was washed with saturated brine ×2, dried over anhydrous magnesium sulfate, and evaporated under reduced pressure to obtain 22 g of Boc aminocaproic acid, which was a transparent viscous liquid at room temperature and solidified at low temperature. Yield: 99.7%.
步骤五：Boc氨基己酸9-十七酯的制备Step 5: Preparation of Boc aminocaproic acid 9-heptadecanate
将9-十七醇(12.5g，48.8mmol)加入反应瓶中，加入Boc氨基辛酸(15.2g，58.6mmol)，加入210ml二氯甲烷，搅拌溶清后加入35ml DIPEA，DMAP(1.19g，9.76mmol)，EDC(23.4g，122mmol)，30℃搅拌反应24小时，TLC监测反应进程，反应完全后，经后处理，柱层析纯化。得Boc氨基辛酸9-十七酯21g，常温下为淡黄色固体。收率：86.5％。Add 9-heptadecanol (12.5g, 48.8mmol) into the reaction bottle, add Boc aminooctanoic acid (15.2g, 58.6mmol), add 210ml dichloromethane, stir and dissolve, then add 35ml DIPEA, DMAP (1.19g, 9.76 mmol), EDC (23.4g, 122mmol), stir and react at 30°C for 24 hours, monitor the reaction progress with TLC, and after the reaction is complete, perform post-treatment and purify by column chromatography. 21g of Boc aminooctanoic acid 9-heptadecanate was obtained, which was a light yellow solid at room temperature. Yield: 86.5%.
步骤六：氨基己酸9-十七酯的制备Step 6: Preparation of 9-heptadecanyl aminocaproate
将Boc氨基己酸9-十七酯5g加入反应瓶中，加入15mL二氯甲烷，搅拌溶清后加入三氟乙酸10mL，在30℃条件下反应3小时，TLC监测反应结束，饱和碳酸钠溶液调pH至碱性，二氯甲烷溶液萃取，无水硫酸镁干燥，减压旋蒸，得到黄色油状物3.74g，收率：94.9％。Add 5g of Boc aminocaproic acid 9-heptadecanate into the reaction bottle, add 15mL of methylene chloride, stir and dissolve, add 10mL of trifluoroacetic acid, and react at 30°C for 3 hours. TLC monitors the end of the reaction and saturated sodium carbonate solution Adjust the pH to alkaline, extract with dichloromethane solution, dry with anhydrous magnesium sulfate, and rotary evaporate under reduced pressure to obtain 3.74g of yellow oil, yield: 94.9%.
步骤七：化合物6的合成Step 7: Synthesis of Compound 6
将氨基己酸9-十七酯(4.1g，11.08mmol)加入反应瓶中，在反应瓶中加入化合物2(2.48g，7.08mmol)，加入DIPEA(2.0g，11.51mmol)，乙醇4mL，在氮气保护的条件下，62℃反应48小时，经TLC检测，到达反应终点，在反应液中加入乙酸乙酯，加入饱和碳酸氢钠溶液洗涤，饱和食盐水洗涤，无水硫酸镁干燥，减压旋蒸，柱层析纯化得到化合物6，3.1g淡黄色液体。收率：68.4％。Add 9-heptadecanyl aminocaproate (4.1g, 11.08mmol) into the reaction flask, add compound 2 (2.48g, 7.08mmol) into the reaction flask, add DIPEA (2.0g, 11.51mmol), and 4 mL of ethanol. Under nitrogen protection, react at 62°C for 48 hours. After TLC detection, the reaction end point is reached. Add ethyl acetate to the reaction solution, add saturated sodium bicarbonate solution to wash, wash with saturated brine, dry over anhydrous magnesium sulfate, and reduce pressure. After rotary evaporation and column chromatography purification, compound 6 was obtained as 3.1 g of light yellow liquid. Yield: 68.4%.
步骤八：化合物lipoND-3的合成Step 8: Synthesis of compound lipoND-3
将化合物6(2.8g，4.38mmol)加入反应瓶中，加入2-溴乙醇(0.82g，6.56mmol)，加入DIPEA(0.85g，6.56mmol)，在氮气保护的条件下62℃反应24小时，经TLC检测，到达反应终点，反应结束，经后处理，柱层析纯化得到lipoND-3，2.12g淡黄色液体。收率：70.8％。Add compound 6 (2.8g, 4.38mmol) into the reaction bottle, add 2-bromoethanol (0.82g, 6.56mmol), add DIPEA (0.85g, 6.56mmol), and react at 62°C for 24 hours under nitrogen protection. After TLC detection, the reaction end point was reached and the reaction was completed. After post-treatment and column chromatography purification, lipoND-3 was obtained as 2.12g of light yellow liquid. Yield: 70.8%.
实施例4：化合物lipoND-4的制备Example 4: Preparation of compound lipoND-4
步骤一至步骤三与实施例1相同Steps 1 to 3 are the same as in Example 1
步骤四：Boc氨基辛酸的制备Step 4: Preparation of Boc aminocaprylic acid
将氨基辛酸(25g，157.04mmol)加入反应瓶中，加入250ml水，搅拌溶清后，加入500ml丙酮。加入Boc酸酐(37.3g，172.74mmol)，三乙胺(31.78g，314.08mmol)，30℃反应4小时。经TLC检测，反应结束，经后处理，得Boc氨基辛酸40.52g，室温下为透明粘稠液体，低温时凝固。收率：99.5％。Add aminocaprylic acid (25g, 157.04mmol) into the reaction bottle, add 250ml of water, stir and dissolve, then add 500ml of acetone. Add Boc acid anhydride (37.3g, 172.74mmol) and triethylamine (31.78g, 314.08mmol), and react at 30°C for 4 hours. After TLC detection, the reaction was completed. After post-processing, 40.52g of Boc aminooctanoic acid was obtained. It was a transparent viscous liquid at room temperature and solidified at low temperature. Yield: 99.5%.
步骤五：Boc氨基辛酸9-十七酯的制备Step 5: Preparation of Boc aminooctanoic acid 9-heptadecanate
将9-十七醇(12.5g，48.8mmol)加入反应瓶中，加入Boc氨基辛酸(15.2g，58.6mmol)，加入210ml二氯甲烷，搅拌溶清后加入35ml DIPEA，DMAP(1.19g，9.76mmol)，EDC(23.4g，122mmol)，30℃搅拌反应24小时，TLC监测反应进程，反应完全后，经后处理，柱层析纯化。得Boc氨基辛酸9-十七酯21g，常温下为淡黄色固体。收率：86.5％。Add 9-heptadecanol (12.5g, 48.8mmol) into the reaction bottle, add Boc aminooctanoic acid (15.2g, 58.6mmol), add 210ml dichloromethane, stir and dissolve, then add 35ml DIPEA, DMAP (1.19g, 9.76 mmol), EDC (23.4g, 122mmol), stir and react at 30°C for 24 hours, monitor the reaction progress with TLC, and after the reaction is complete, perform post-treatment and purify by column chromatography. 21g of Boc aminooctanoic acid 9-heptadecanate was obtained, which was a light yellow solid at room temperature. Yield: 86.5%.
步骤六：氨基辛酸9-十七酯的制备Step 6: Preparation of 9-heptadecanyl aminooctanoate
将Boc氨基辛酸9-十七酯19.7g加入反应瓶中，加入60mL二氯甲烷，搅拌溶清后加入三氟乙酸40mL，在30℃条件下反应3小时，TLC监测反应结束，经后处理，得到黄色油状物15g，收率：95.2％。Add 19.7g of Boc 9-heptadecayl aminooctanoate into the reaction bottle, add 60mL of dichloromethane, stir and dissolve, add 40mL of trifluoroacetic acid, and react at 30°C for 3 hours. TLC monitors the end of the reaction. After post-processing, 15 g of yellow oil was obtained, yield: 95.2%.
步骤七：化合物9的合成Step 7: Synthesis of Compound 9
将氨基辛酸9-十七酯(5g，12.57mmol)加入反应瓶中，在反应瓶中加入化合物M3(2.65g，7.54mmol)，加入DIPEA(2.23g，17.56mmol)，乙醇5mL，在氮气保护的条件下，62℃反应48小时，经TLC检测，到达反应终点，经后处理，柱层析纯化得到化合物9，3.5g，淡黄色液体。收率：67.7％。Add 9-heptadecanyl aminooctanoate (5g, 12.57mmol) into the reaction flask, add compound M3 (2.65g, 7.54mmol) into the reaction flask, add DIPEA (2.23g, 17.56mmol), and 5 mL of ethanol, under nitrogen protection Under the conditions, the reaction was carried out at 62°C for 48 hours. After TLC detection, the reaction end point was reached. After post-treatment and purification by column chromatography, compound 9, 3.5 g, was obtained as a light yellow liquid. Yield: 67.7%.
步骤八：化合物lipoND-4的合成Step 8: Synthesis of compound lipoND-4
将化合物9加入反应瓶中，加入2-溴乙醇，加入DIPEA，在氮气保护的条件下62℃反应24小时，经TLC检测，到达反应终点，反应结束，向反应液中加入乙酸乙酯，饱和碳酸氢钠溶液洗涤，饱和食盐水洗涤，无水硫酸镁干燥，减压旋蒸，柱层析纯化得到化合物lipoND-4，收率：72.2％。Add compound 9 to the reaction bottle, add 2-bromoethanol, add DIPEA, and react at 62°C for 24 hours under nitrogen protection. After TLC detection, the reaction end point is reached and the reaction is completed. Add ethyl acetate to the reaction solution and make it saturated. Washed with sodium bicarbonate solution, washed with saturated brine, dried over anhydrous magnesium sulfate, evaporated under reduced pressure, and purified by column chromatography to obtain compound lipoND-4, yield: 72.2%.
实施例5：化合物lipoND-5的制备Example 5: Preparation of compound lipoND-5
步骤一至步骤三与实施例1相同Steps 1 to 3 are the same as in Example 1
步骤四：Boc氨基辛酸的制备Step 4: Preparation of Boc aminocaprylic acid
将氨基辛酸(25g，157.04mmol)加入反应瓶中，加入250ml水，搅拌溶清后，加入500ml丙酮。加入Boc酸酐(37.3g，172.74mmol)，三乙胺(31.78g，314.08mmol)，30℃反应4小时。经TLC检测，反应结束，经后处理，得Boc氨基辛酸40.52g，室温下为透明粘稠液体，低温时凝固。收率：99.5％。Add aminocaprylic acid (25g, 157.04mmol) into the reaction bottle, add 250ml of water, stir and dissolve, then add 500ml of acetone. Add Boc acid anhydride (37.3g, 172.74mmol) and triethylamine (31.78g, 314.08mmol), and react at 30°C for 4 hours. After TLC detection, the reaction was completed. After post-processing, 40.52g of Boc aminooctanoic acid was obtained. It was a transparent viscous liquid at room temperature and solidified at low temperature. Yield: 99.5%.
步骤五：Boc氨基辛酸9-十七酯的制备Step 5: Preparation of Boc aminooctanoic acid 9-heptadecanate
将9-十七醇(12.5g，48.8mmol)加入反应瓶中，加入Boc氨基辛酸(15.2g，58.6mmol)，加入210ml二氯甲烷，搅拌溶清后加入35ml DIPEA，DMAP(1.19g，9.76mmol)，EDC(23.4g，122mmol)，30℃搅拌反应24小时，TLC监测反应进程，反应完全后，经后处理，柱层析纯化。得Boc氨基辛酸9-十七酯21g，常温下为淡黄色固体。收率：86.5％。Add 9-heptadecanol (12.5g, 48.8mmol) into the reaction bottle, add Boc aminooctanoic acid (15.2g, 58.6mmol), add 210ml dichloromethane, stir and dissolve, then add 35ml DIPEA, DMAP (1.19g, 9.76 mmol), EDC (23.4g, 122mmol), stir and react at 30°C for 24 hours, monitor the reaction progress with TLC, and after the reaction is complete, perform post-treatment and purify by column chromatography. 21g of Boc aminooctanoic acid 9-heptadecanate was obtained, which was a light yellow solid at room temperature. Yield: 86.5%.
步骤六：氨基辛酸9-十七酯的制备Step 6: Preparation of 9-heptadecanyl aminooctanoate
将Boc氨基辛酸9-十七酯19.7g加入反应瓶中，加入60mL二氯甲烷，搅拌溶清后加入三氟乙酸40mL，在30℃条件下反应3小时，TLC监测反应结束，经后处理，得到黄色油状物15g，收率：95.2％。Add 19.7g of Boc 9-heptadecayl aminooctanoate into the reaction bottle, add 60mL of dichloromethane, stir and dissolve, add 40mL of trifluoroacetic acid, and react at 30°C for 3 hours. TLC monitors the end of the reaction. After post-processing, 15 g of yellow oil was obtained, yield: 95.2%.
步骤七：化合物11的合成Step 7: Synthesis of Compound 11
将氨基辛酸9-十七酯(5g，12.57mmol)加入反应瓶中，在反应瓶中加入化合物M1(2.55g，7.54mmol)，加入DIPEA(2.23g，17.56mmol)，乙醇5mL，在氮气保护的条件下，62℃反应48小时，经TLC检测，到达反应终点，经后处理，柱层析纯化得到化合物11，淡黄色液体。收率：65.7％。Add 9-heptadecanyl aminooctanoate (5g, 12.57mmol) into the reaction flask, add compound M1 (2.55g, 7.54mmol) into the reaction flask, add DIPEA (2.23g, 17.56mmol), and 5 mL of ethanol, under nitrogen protection Under the conditions of 62°C, the reaction was carried out for 48 hours. After TLC detection, the reaction end point was reached. After post-treatment and purification by column chromatography, compound 11 was obtained as a light yellow liquid. Yield: 65.7%.
步骤八：化合物lipoND-5的合成Step 8: Synthesis of compound lipoND-5
将化合物11加入反应瓶中，加入2-溴乙醇，加入DIPEA，在氮气保护的条件下62℃反应24小时，经TLC检测，到达反应终点，反应结束，向反应液中加入乙酸乙酯，饱和碳酸氢钠溶液洗涤，饱和食盐水洗涤，无水硫酸镁干燥，减压旋蒸，柱层析纯化,得到化合物lipoND-5，收率：65％。Add compound 11 to the reaction bottle, add 2-bromoethanol, add DIPEA, and react at 62°C for 24 hours under nitrogen protection. After TLC detection, the reaction end point is reached and the reaction is completed. Add ethyl acetate to the reaction solution and make it saturated. Wash with sodium bicarbonate solution, wash with saturated brine, dry over anhydrous magnesium sulfate, evaporate under reduced pressure, and purify by column chromatography to obtain compound lipoND-5, yield: 65%.
比较例1：采用市售lipid5(Moderna研发的COVID-19疫苗mRNA-1273阳离子脂质递送载体的研发代号)。Comparative Example 1: Commercially available lipid5 (the research and development code name of the COVID-19 vaccine mRNA-1273 cationic lipid delivery vector developed by Moderna) was used.
比较例2：采用市售SM-102(Moderna研发的COVID-19疫苗mRNA-1273阳离子脂质递送载体的研发代号)。Comparative Example 2: Commercially available SM-102 (the research and development code name of the COVID-19 vaccine mRNA-1273 cationic lipid delivery vector developed by Moderna) was used.
安全性及递送效率评价Safety and delivery efficiency evaluation
采用微流控混合技术制备LNPPreparation of LNPs using microfluidic mixing technology
有机相配置：将可电离阳离子脂质、DSPC、胆固醇和DMG-PEG2000以50:10:38.5:1.5的摩尔比10mM的浓度溶于无水乙醇中，制备有机相。Organic phase configuration: Dissolve ionizable cationic lipids, DSPC, cholesterol and DMG-PEG2000 in absolute ethanol at a molar ratio of 10mM: 50:10:38.5:1.5 to prepare the organic phase.
水相配置：2mol/L冰醋酸的配制，将6mL冰醋酸稀释至50mL，即得2mol/L冰醋酸。取2mol/L冰醋酸溶液20.5ml，醋酸钠1.22g，加水稀释至1L，摇匀，即得pH＝4的50mM柠檬酸缓冲液的缓冲液。用50mM柠檬酸缓冲液溶解siRNA得到水相。Aqueous phase configuration: To prepare 2mol/L glacial acetic acid, dilute 6mL glacial acetic acid to 50mL to obtain 2mol/L glacial acetic acid. Take 20.5ml of 2mol/L glacial acetic acid solution and 1.22g of sodium acetate, dilute to 1L with water, and shake well to obtain a 50mM citric acid buffer with pH=4. Dissolve siRNA in 50mM citrate buffer to obtain an aqueous phase.
随后，将两相溶液吸入注射器，然后将注射器出口和样品导入管连接，并固定在注射泵上。使用RSPO-BD注射器泵(Biotaor instrument,Jiaxin)将水相和有机相混合。有机相设置流速为1mL/min，水相流速为3mL/min，总流速为4mL/min。制备后使用分子量为10kDa截止分子量的透析袋对在1×PBS进行持续4小时的透析。然后通过0.22μm过滤器将所得溶液过滤并储存于4℃。Subsequently, the two-phase solution is sucked into the syringe, and then the syringe outlet and the sample introduction tube are connected and fixed on the syringe pump. The aqueous and organic phases were mixed using an RSPO-BD syringe pump (Biotaor instrument, Jiaxin). The flow rate of the organic phase was set to 1mL/min, the flow rate of the aqueous phase was 3mL/min, and the total flow rate was 4mL/min. After preparation, dialysis was performed in 1×PBS for 4 hours using a dialysis bag with a molecular weight cutoff of 10 kDa. The resulting solution was then filtered through a 0.22 μm filter and stored at 4°C.
实验例1：TNS法测量LNP的pKa值Experimental example 1: TNS method to measure the pKa value of LNP
纳米颗粒的表观pKa是预测纳米颗粒包裹RNA效率的可靠标准，纳米粒的表观pKa与其疗效和毒性有很高的相关性。TNS荧光法对LNP的pKa值的测量非常灵敏，已被广泛用于测量LNP的pKa。TNS带负电荷，当与带正电荷的LNP结合时，产生荧光增强效果。随着pH值的降低，TNS和可电离表面的相互作用增加，导致了荧光的稳定增加。在特定的pH值下，所有的可电离基团带电荷，荧光值达到最大，pKa值被定义为产生半最大荧光强度的pH值。The apparent pKa of nanoparticles is a reliable criterion for predicting the efficiency of nanoparticles in encapsulating RNA. The apparent pKa of nanoparticles is highly correlated with their efficacy and toxicity. The TNS fluorescence method is very sensitive for measuring the pKa value of LNP and has been widely used to measure the pKa value of LNP. TNS is negatively charged and produces a fluorescence enhancement effect when combined with positively charged LNPs. As the pH decreases, the interaction between TNS and the ionizable surface increases, resulting in a steady increase in fluorescence. At a specific pH value, all ionizable groups are charged and the fluorescence value reaches a maximum. The pKa value is defined as the pH value that produces half-maximal fluorescence intensity.
具体操作如下：The specific operations are as follows:
配置缓冲液：称取氯化钠7.6g，乙酸铵0.77g，4-羟乙基哌嗪乙磺酸(Hepes)2.383g，2-(N-吗啡啉)乙磺酸(MES)2.13g于1L纯净水中溶解，用0.1M HCl和0.1M NaOH以0.5的步长滴定至pH值从3-10，每个pH值取3mL放入PE管中。Prepare buffer: Weigh 7.6g of sodium chloride, 0.77g of ammonium acetate, 2.383g of 4-hydroxyethylpiperazineethanesulfonic acid (Hepes), and 2.13g of 2-(N-morpholine)ethanesulfonic acid (MES). Dissolve in 1L pure water, titrate with 0.1M HCl and 0.1M NaOH in steps of 0.5 to a pH value from 3-10, and put 3mL of each pH value into a PE tube.
取40μL 10mmol/L制得的LNP加入由上述方法制得的不同pH值的缓冲液中。称取3.5mg 2-硝基苯胺-4,4,5,5-四甲酸(TNS)于25mL二甲基亚砜(DMSO)中溶解，浓度约为400μmol。将100μL的TNS溶液与3mL的LNP混合，激发和发射波长分别为391nm和436nm，荧光数据采用s型最佳拟合分析。Take 40 μL of the LNP prepared at 10 mmol/L and add it to the buffers with different pH values prepared by the above method. Weigh 3.5 mg of 2-nitroaniline-4,4,5,5-tetracarboxylic acid (TNS) and dissolve it in 25 mL of dimethyl sulfoxide (DMSO). The concentration is approximately 400 μmol. 100 μL of TNS solution was mixed with 3 mL of LNP. The excitation and emission wavelengths were 391 nm and 436 nm, respectively. The fluorescence data was analyzed using s-type best fitting.
从TNS法测量LNP的pKa值(见附图6-图11)的结果可知，本发明所制备的化合物pKa均在6.5～6.7之间，与SM-102和lipid 5的pKa值相似，说明在疏水尾部加入醚键可电离脂质会提高LNP递送siRNA的效率，符合可电离阳离子脂质的设计策略。From the results of measuring the pKa value of LNP by the TNS method (see Figures 6 to 11), it can be seen that the pKa values of the compounds prepared by the present invention are all between 6.5 and 6.7, which are similar to the pKa values of SM-102 and lipid 5, indicating that in Adding ether bond ionizable lipids to the hydrophobic tail will improve the efficiency of LNP delivery of siRNA, which is in line with the design strategy of ionizable cationic lipids.
实验例2：CCK-8法评估LNP对于NIH-3T3和HSC-T6的安全性Experimental Example 2: CCK-8 method to evaluate the safety of LNP for NIH-3T3 and HSC-T6
HSC-T6和NIH-3T3细胞系均购自中国武汉普诺赛生命科学技术有限公司，使用10％胎牛血清或小牛血清、100U/mL青霉素和100μg/mL链霉素的高糖DMEM中进行培养，放置在5％ CO2的湿化气氛下，37℃的培养箱中。HSC-T6 and NIH-3T3 cell lines were purchased from Wuhan Pronosai Life Science Technology Co., Ltd., China, and maintained in high-glucose DMEM using 10% fetal bovine serum or calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. For culture, place in a 37 °C incubator in a humidified atmosphere of 5%CO2 .
1)细胞毒性研究按照CCK-8试剂盒的步骤，将HSC-T6和NIH-3T3以每孔5×103的密度在96孔板中培养12h。1) Cytotoxicity study: According to the steps of the CCK-8 kit, HSC-T6 and NIH-3T3 were cultured in a 96-well plate at a density of 5 × 103 per well for 12 hours.
2)将不同脂质浓度的LNPs(10、50、100、150、200、500μM)与细胞孵育24h。2) Incubate LNPs with different lipid concentrations (10, 50, 100, 150, 200, 500 μM) with cells for 24 h.
3)孵育结束后通过添加10％CCK-8检测试剂用酶标仪测定450nm处的吸光度值。以未处理细胞的活力作为对照。细胞存活率按照下式进行计算：3) After the incubation, add 10% CCK-8 detection reagent and measure the absorbance value at 450nm with a microplate reader. The viability of untreated cells was used as a control. Cell survival rate is calculated according to the following formula:
细胞存活率＝[(As-Ab)/(Ac-Ab)]×100％Cell survival rate=[(As -Ab )/(Ac -Ab )]×100%
As:实验孔吸光度(含细胞、培养基、CCK-8溶液和药物溶液)；As: absorbance of experimental wells (including cells, culture medium, CCK-8 solution and drug solution);
Ac:对照孔吸光度(含细胞、培养基、CCK-8溶液，不含药物)；Ac: absorbance of control well (including cells, culture medium, CCK-8 solution, excluding drugs);
Ab:空白孔吸光度(含培养基、CCK-8溶液，不含细胞、药物)。Ab: Absorbance of blank wells (including culture medium, CCK-8 solution, but not cells or drugs).
从CCK-8法评估LNP对于NIH-3T3和HSC-T6的安全性的实验结果(见附图12)可知，显示每种化合物即使在最高浓度(500μM)下两种细胞的存活力都在80％以上，说明lipid5，SM-102和lipoND系列的化合物均没有显著的细胞毒性，因此证明化合物I醚键的加入不会对LNPs产生任何额外的毒性，制备的LNP具有良好的生物安全性和生物相容性。From the experimental results of the CCK-8 method to evaluate the safety of LNP for NIH-3T3 and HSC-T6 (see Figure 12), it can be seen that the viability of both cells of each compound is 80 even at the highest concentration (500 μM). % or above, indicating that the lipid5, SM-102 and lipoND series of compounds have no significant cytotoxicity. Therefore, it is proved that the addition of the ether bond of compound I will not cause any additional toxicity to LNPs, and the prepared LNPs have good biosafety and biological properties. compatibility.
实验例3：载HSP47 siRNA的LNP对于NIH-3T3细胞的转染率Experimental Example 3: Transfection rate of HSP47 siRNA-loaded LNP into NIH-3T3 cells
将NIH-3T3细胞接种于6孔板(每孔3×105个细胞)，在含10％ CS和1％ S/P的DMEM培养基中孵育。次日，将细胞培养基更换为无血清培养基。然后将HSP47 siRNA和装载HSP47siRNA的LNPs加入细胞中。在37℃的CO2培养箱中培养细胞5-6小时，再更换含有血清的培养基并培养细胞48小时。NIH-3T3 cells were seeded in 6-well plates (3×105 cells per well) and incubated in DMEM medium containing 10% CS and 1% S/P. The next day, the cell culture medium was replaced with serum-free medium. HSP47 siRNA and HSP47 siRNA-loaded LNPs were then added to the cells. Culture the cells in aCO2 incubator at 37°C for 5-6 hours, then replace the medium containing serum and culture the cells for 48 hours.
按照RNA提取试剂盒Cell/Tissue TotalRNA Isolation Kit V2(Vazyme,Nanjing)建立的流程提取细胞总RNA。首先加入500μL Buffer RL裂解样本。然后转移裂解产物至FastPure gDNA-Filter Column lll,12,000rpm离心30s，收集滤液。加入0.5倍滤液体积无水乙醇至滤液，充分混匀。转移全部混合物至FastPure RNA Column lll,12,000rpm离心30s，弃滤液。加入700μL Buffer RW1，12,000rpm离心30s，弃滤液。加入700μL Buffer RW2(已加入无水乙醇)，12,000pm离心30sec，弃滤液。加入500μL Buffer RW2(已加入无水乙醇)，12,000rpm离心2min，弃收集管。最后加入50-200μL RNase-free ddH2O，12,000rpm离心1min得到总RNA。再按照下列条件进行One Step qRT-PCR反应：Follow the RNA extraction kit Total cellular RNA was extracted using the process established by Cell/Tissue TotalRNA Isolation Kit V2 (Vazyme, Nanjing). First add 500μL Buffer RL to lyse the sample. Then transfer the lysate to FastPure gDNA-Filter Column lll, centrifuge at 12,000 rpm for 30 s, and collect the filtrate. Add 0.5 times the volume of filtrate absolute ethanol to the filtrate and mix thoroughly. Transfer the entire mixture to FastPure RNA Column lll, centrifuge at 12,000 rpm for 30 s, and discard the filtrate. Add 700 μL Buffer RW1, centrifuge at 12,000 rpm for 30 s, and discard the filtrate. Add 700 μL Buffer RW2 (absolute ethanol has been added), centrifuge at 12,000pm for 30 seconds, and discard the filtrate. Add 500 μL Buffer RW2 (absolute ethanol has been added), centrifuge at 12,000 rpm for 2 minutes, and discard the collection tube. Finally, add 50-200 μL RNase-free ddH2 O, and centrifuge at 12,000 rpm for 1 min to obtain total RNA. Then perform the One Step qRT-PCR reaction according to the following conditions:
从载HSP47 siRNA的LNP对于NIH-3T3细胞的转染率结果(见附图13)可知，qRT-PCR实验结果显示LNPs对NIH-3T3细胞中的HSP47基因的沉默效果与空白组和naked siRNA组相比均有显著性差异。其中，SM-102制备的LNPs的基因沉默效率为58.44±0.04％，lipid5制备的LNPs的基因沉默效率约为15.45±0.08％。而在lipoND系列化合物中，lipoND-1，lipoND-2和lipoND-4所制备的LNPs的沉默效率分别为61.76±0.07％，54.65±0.06％和62.30±0.06％，lipoND-1和lipoND-4的基因沉默效率优于SM-102和lipid5。lipoND-2的基因沉默效率与SM-102相近，优于lipid5。从结构上看lipoND-1，lipoND-2和lipoND-4三种阳离子脂质与lipid5相对比，进行了改造，lipoND系列化合物中均含有醚键，说明将醚键加入阳离子脂质中的合适位置可以显著改善LNP的递送效率。From the transfection rate results of HSP47 siRNA-loaded LNPs on NIH-3T3 cells (see Figure 13), it can be seen that the qRT-PCR experimental results show that the silencing effect of LNPs on the HSP47 gene in NIH-3T3 cells is different from that of the blank group and naked siRNA group. There are significant differences in comparison. Among them, the gene silencing efficiency of LNPs prepared by SM-102 was 58.44±0.04%, and the gene silencing efficiency of LNPs prepared by lipid5 was approximately 15.45±0.08%. Among the lipoND series compounds, the silencing efficiencies of LNPs prepared by lipoND-1, lipoND-2 and lipoND-4 were 61.76±0.07%, 54.65±0.06% and 62.30±0.06% respectively. The silencing efficiencies of lipoND-1 and lipoND-4 The gene silencing efficiency is better than SM-102 and lipid5. The gene silencing efficiency of lipoND-2 is similar to SM-102 and better than lipid5. From a structural point of view, the three cationic lipids lipoND-1, lipoND-2 and lipoND-4 have been modified compared with lipid5. LipoND series compounds all contain ether bonds, indicating the appropriate position for adding ether bonds to cationic lipids. The delivery efficiency of LNP can be significantly improved.
实验例4：载Cy5-siRNA的LNP对于肝脏的靶向性研究Experimental Example 4: Study on liver targeting of LNP loaded with Cy5-siRNA
将负载Cy5-siRNA的LNPs(浓度为0.5mg/kg siRNA)静脉注射给每只小鼠，24h处死后，对主要器官进行离体成像(IVIS Lumina 3,PerkinElmer)。24h后，对心脏、肝脏、脾脏、肺脏和肾脏等主要器官进行体外成像。LNPs loaded with Cy5-siRNA (concentration of 0.5 mg/kg siRNA) were intravenously injected into each mouse. After 24 hours of sacrifice, the main organs were imaged ex vivo (IVIS Lumina 3, PerkinElmer). After 24 hours, in vitro imaging was performed on major organs such as the heart, liver, spleen, lungs, and kidneys.
从载Cy5-siRNA的LNP对于肝脏的靶向性研究结果(见附图14)可知，LipoND-1组肝脏荧光强度为lipid 5组的1.3倍。LipoND-4组肝脏荧光强度为lipid 5组的1.1倍。Cy5-siRNA组肾组织可见明显荧光信号，证实成功验证了siRNA分子的肾脏快速清除。相反，在其他器官中观察到极少量来自LNP的荧光信号，这表明在脂质尾部加入醚键后lipid 5的肝脏靶向性未受影响。各组中，LipoND-1和LipoND-4的肝脏靶向性最强。From the results of the liver targeting study of Cy5-siRNA-loaded LNP (see Figure 14), it can be seen that the liver fluorescence intensity of the LipoND-1 group was 1.3 times that of the lipid 5 group. The liver fluorescence intensity of the LipoND-4 group was 1.1 times that of the lipid 5 group. Obvious fluorescence signals were visible in the kidney tissue of the Cy5-siRNA group, confirming the successful renal clearance of siRNA molecules. In contrast, minimal fluorescence signals from LNPs were observed in other organs, suggesting that the liver targeting of lipid 5 was unaffected upon the addition of ether bonds to the lipid tail. Among each group, LipoND-1 and LipoND-4 have the strongest liver targeting properties.
综上所述，本发明所合成出来的新型阳离子脂质化合物，通过在疏水尾部加入醚键，实现对LNP递送siRNA的效率的提高，细胞实验证实了在疏水尾部加入醚键并不会使化合物产生毒性。同时，设计合成了多种可电离阳离子脂质，可用于与其他脂质组分(磷脂、胆固醇和PEG脂质)结合，以便制备包载核酸的脂质纳米粒，从而促进体外和体内的核酸递送，并通过实验验证了其生物安全性和高效的siRNA转染能力。In summary, the new cationic lipid compound synthesized by the present invention can improve the efficiency of LNP delivery of siRNA by adding ether bonds to the hydrophobic tail. Cell experiments have confirmed that adding ether bonds to the hydrophobic tail does not make the compound produce toxicity. At the same time, a variety of ionizable cationic lipids have been designed and synthesized, which can be used to combine with other lipid components (phospholipids, cholesterol and PEG lipids) to prepare lipid nanoparticles encapsulating nucleic acids, thereby promoting the synthesis of nucleic acids in vitro and in vivo. delivery, and experimentally verified its biosafety and efficient siRNA transfection capabilities.
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be equivalently replaced; and these modifications or substitutions do not deviate from the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention. scope.
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| CN110520409A (en)* | 2017-03-15 | 2019-11-29 | 摩登纳特斯有限公司 | Compound and composition for Intracellular delivery therapeutic agent |
| CN113264842A (en)* | 2021-07-21 | 2021-08-17 | 江苏普瑞康生物医药科技有限公司 | Lipid compound, lipid carrier containing same, nucleic acid lipid nanoparticle composition and pharmaceutical preparation |
| CN114044741A (en)* | 2022-01-13 | 2022-02-15 | 北京悦康科创医药科技股份有限公司 | Cationic lipid compound, composition containing same and application |
| CN114763324A (en)* | 2021-01-15 | 2022-07-19 | 纳肽得(青岛)生物医药有限公司 | Lipid compound, liposome and pharmaceutical composition |
| CN114773217A (en)* | 2022-06-20 | 2022-07-22 | 深圳市瑞吉生物科技有限公司 | Cationic lipid compounds and compositions for delivery of nucleic acids and uses |
| WO2023178065A2 (en)* | 2022-03-15 | 2023-09-21 | Broadpharm Inc. | Lipid compounds and lipid nanoparticles |
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| CN110520409A (en)* | 2017-03-15 | 2019-11-29 | 摩登纳特斯有限公司 | Compound and composition for Intracellular delivery therapeutic agent |
| CN114763324A (en)* | 2021-01-15 | 2022-07-19 | 纳肽得(青岛)生物医药有限公司 | Lipid compound, liposome and pharmaceutical composition |
| CN113264842A (en)* | 2021-07-21 | 2021-08-17 | 江苏普瑞康生物医药科技有限公司 | Lipid compound, lipid carrier containing same, nucleic acid lipid nanoparticle composition and pharmaceutical preparation |
| CN114044741A (en)* | 2022-01-13 | 2022-02-15 | 北京悦康科创医药科技股份有限公司 | Cationic lipid compound, composition containing same and application |
| WO2023178065A2 (en)* | 2022-03-15 | 2023-09-21 | Broadpharm Inc. | Lipid compounds and lipid nanoparticles |
| CN114773217A (en)* | 2022-06-20 | 2022-07-22 | 深圳市瑞吉生物科技有限公司 | Cationic lipid compounds and compositions for delivery of nucleic acids and uses |
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