Water-soluble liposomes and lipopeptides for nucleic acid delivery
Water-soluble liposomes and lipopeptides for nucleic acid delivery
Water-soluble lipopolymers and lipopeptides are a class of nonviral gene vectors that combine the increased permeability of lipid materials to cell membranes by D N A with the condensation and merging of polycations with D N A to promote gene escape in introns. Lipid macromolecules were prepared by the chemical reaction of cholesteryl acetate chloroformate with primary or secondary amines of branched polyethyleneimine (P E I ) with a molecular mass of 1800 D a . Lipopeptides were prepared by mixing C K i V -succinimidyl)-iV, N , A T , J V -tetramethyluronium tetrafluoroborate (T S T U ) with struvitecholic acid in the presence of an excess of diisopropylethylamine (D I P E A ) and reacting it with peptides derived from human fish spermatoglycerin. The use of P E I with a molecular mass of 1800 D a is intended to avoid toxicity caused by high molecular mass P E I . Author: T. Friedman et al., Translated by W. Jing et al. This experiment is from "Gene Transfer".
Operation method
Preparation and use of water-soluble lipopolymers/pDNA and lipopeptide/pDNA complexes Move Preparation and use of water-soluble lipopolymers/pDNA and lipopeptide/pDNA complexes Materials reagents Important: All solvents are HPLC grade. Branched Polyethylenimine (PEI; 1800Da; Polysciences) C T-26 colon cancer cells Cholesteryl chloroformate diethyl ether Diisopropylethylamine solution (DIPEA; l m o l /L in DMF) Dimethylformamide (D M F ) Ethyl bromide (0.5 ug/ml in 1X T B E ) Fetal Bovine Serum (FBS) 5 % glucose injection Hydrochloric acid (H C 1; 0. lm o l/L ) lithocholic acid methanol dichloromethane Mouse (5 weeks old BAXJB/c; Simonsen Laboratories) Phosphate buffer (P B S ; tissue culture grade) REPORTED GENE: C M V promoter-driven plasmid encoding fluorokinase D N A ( p C M V _Luc) R P M I 1640 Tissue culture medium Therapeutic gene: p2C M V m I L -12, encoding mouse i l - 2 subunits P3 5 and p4 0 , transcriptionally controlled by separate C M C promoters, respectively Triethylamine Trifluoroacetic acid (TFA; 95% vs. 5%) Tris-Boric Acid-EDTA Buffer (TBE; commercially available or formulated, see Sambrookan d Rus_sell 2001) O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU) solution (l m o l / L in DMF) Apparatus Agarose gel electrophoresis equipment Bicinchoninic acid (B C A ) total protein quantification kit (Pierce) m I L -12 p70 ELISA kit (E L I S A ) (Pharmingen) High Performance Liquid Chromatograph (H P L C ) including C 18 column (V y d a c ) luminometer (Dynex Technologies) Matrix-assisted laser desorption ionization-time of flight mass spectrometry (M A LD I-T O F ) ( ^ PerspectiveVoyagerD E S T R , Applied Biosystems) Nuclear Magnetic Resonance Spectrometer (N M R ) (Varian) Peptide synthesizer (e.g. Applied Biosystems 433A peptide synthesizer) and related equipment Standard tissue culture equipment and ancillary facilities Zeta Potential Analyzer (ZETA PALS, Brookhaven Instruments) METHODOLOGY Synthesis and characterization of water-soluble lipopolymers 1 . 3 g of PEI (1800D a) was dissolved with IOOmI triethylamine in IOml anhydrous dichloromethane and stirred in an ice bath for 30 min. 2. I g of cholesteryl chloroformate was dissolved in 5 ml of cold dichloromethane, slowly added to the above solution of PEI, and stirred in an ice-water bath for 12 h. The product was dried, dissolved in 50 ml of 0.1mol/L HCl solution, and filtered. 3. Extract the above aqueous solution with 100 ml of dichloromethane and filter, concentrate the filtrate, precipitate in a large amount of acetone, dry. Wash with methanol and ether. 4 . The molecular mass of the product was verified by MALDI-T0 F , using trans-4-hydroxy-3-methoxycinnamic acid as substrate. The structure was verified by 1 H NMR. The water-soluble lipopolymers can be stored at 1 20°C for 6 months. Synthesis and characterization of water-soluble lipopeptides For more product details, please visit Aladdin Scientific website.
![5 . 合成以下脂肤： His-His-T y r -A r g -A r g -A r g -His-C y s-Ser-A r g -A r g -A r g -L e u -His~H i s 。 这一序列对应于人鱼精蛋白51〜6 3 的氨基酸残基，除了以赖氨酸代替5 7 位半胱氨 酸外，另外在氨基端与羧基端增加了组氨酸残基。肽的合成使用标准的F m o c 固相 合成法，以 2, 2, 4, 6, 7-五甲基二氢苯并呋喃-5-横 酰 基 （2 , 2 , 4 ， 6， 7卞6加3111- ethyldihydrobenzofurane~5-sulfonyl， P f p) 保护精氨酸的侧基，以 三 苯 甲 基 （T r t) 保护组氨酸侧基， 1-(4， 4-二甲基-2， 6-二氧杂亚环己基）3-甲 基 丁 基 [1-(4， 4- dimehyl-2, 6-dioxocyclohexylidene)3-methylbutyl， D d e ] 保护赖氨酸侧基。用叔丁 氧 羰 基 （t-B o c) 保护其他氨基酸。 6•用5 % 的 T F A 处理固定于树脂上的合成多肽，使赖氨酸的e■氨基从D d e 脱保护。用 1 0 0 % 甲醇洗几次。这一步骤得到约50M m o l 的多肽，并进行下一步反应。 7•将lOOpmol/L TSTU溶 液 （lmol/L 溶 于 DM F中）逐步滴入到含3 倍摩尔质量的 DIPEA (lmol/L 溶 于 DMF中）的 IOOmI 的石胆酸溶液中（l m〇 l/L 溶 于 DM F中）， 室温下轻摇2h。 8 . 将步骤6 中带有肽的树脂加入到 T S T U /石胆酸/DIPEA 混合溶液中，室温下轻 摇 12h 。](http://img.dxycdn.com/trademd/upload/userfiles/image/2016/08/A1470389694056qciz9q4jwjpng_small.jpg)
![9 . 用 D M F 洗涤反应物。室温下用9 5 % 的 T F A 处 理 90m i n 以使肽从树脂上断裂下来。 以 I O O O g 的转速离心分离树脂。利用带反相C 18柱 的 H P L C 分离纯化肽。通过氨基 酸分析确认肽的组成，并 利 用 M A L D I - T O F 质谱测定其分子质量，测量酪氨酸在 274. 5n m 的吸光值确定肽的浓度[ e274.5 = 14： 00L / (m o l . c m )]。 脂高分子/pDNA及脂肽/pDNA/复合物的制备 10•在5 % 的葡萄糖溶液中，以 1〜2 5 的不同N / P 比，混 合 p D N A 与 脂 高 分 子 （或脂 肽） ，室温下静置15〜20m i n ，以制备脂高分子/ p D N A (或脂肽/p D N A ) 复合物。 N / P 比是指水溶性脂髙分子及脂肽中N 原子数与质粒D N A 中磷原子的比例。 11. 于 I X T B E 中用琼脂糖凝胶电泳分析上述复合物。以 O.Sfig/m l 的溴化乙锭进行染 色，并 在 U V 灯下观察。 12. 用 Z e t a P A L S 测量复合物的平均粒径及电势。 体外转染及萤光蛋白酶活力分析 13•在37°C ， 5 % C 0 2 培养箱中，用 含 10% F B S 的 R P M I 1640培养基培养C T -26结肠 癌细胞。 14.在 6 孔板中以每孔3X 105 的密度接种C T -26细胞，使用含10% F B S 的 R P M I 1640 培养基。在细胞达到7 0 % 密度时，用新制的脂高分子/p D N A 或脂肽/p D N A 复合物 转 染 C T -26细胞，剂量为2.5M g /孔 ，使用无血清的培养基。 15•将复合物与细胞于37°C 、 C O 2 培养箱中培养6h 后，用 2m l 完全培养基替换旧培养 基 ，并继续于37°C 培养36h 。 16. 用 P B S 洗细胞。裂解细胞，用 B C A 总蛋白质定量试剂盒测定蛋白质总量。 17. 用 I u m i n ometer测定萤光素酶的活力，并以蛋白质总量归一化R L U 值 。 瘤内基因传输 18. 在5周龄6八1^/(；小鼠左侧皮下注射以106个(：丁-26细胞以成瘤。 10〜15€ 1后，肿 瘤体积为100〜120m m 3。 19. 以 每 只 荷 瘤 小 鼠 25鸿 质 粒 D N A 的剂量瘤内注射5〇 4新鲜制备的脂高分子/ p2C M V m I L -12 或脂肽/p2C M V m I L -12 复合物。注射 P2C M V m I L -12 裸 D N A 及 5% 葡萄糖作为对照。 20. 单次瘤内注射后，观测小鼠肿瘤生长情况。在 48d 内，记录肿瘤体积的变化。 21. 注射48h 后分离肿瘤组织，将肿瘤组织切成小片， 37°C培养24h ，通过 E L I S A 分析 培养液中m I L -12 p 7 0 的浓度。](http://img.dxycdn.com/trademd/upload/userfiles/image/2016/08/A1470389702932mw6e87s8ufpng_small.jpg)