lipoplex and LPD nanoparticles for in vivo gene delivery
lipoplex and LPD nanoparticles for in vivo gene delivery
The core of gene therapy research lies in the development of suitable delivery systems. Although most clinical experiments currently utilize viral carrier systems, cationic liposomes, because of their safety and versatility, are promising gene carriers for a wide range of applications. Viral vectors may cause genetic mutations and carcinogenesis, and repeated injections of viral vectors induce an immune response that weakens transgene expression. It is because of this limitation of virus carriers that cationic liposomes have shown a distinct advantage. Although cationic liposomes are less efficient at transfection than virus carriers, their safety, versatility, ease of preparation, low immunogenicity, and good biocompatibility have led to their widespread use in both laboratory and clinical trials. Author: T. Friedman et al, Translator: Jingwei et al, This experiment is from "Gene Transfer".
Operation method
Preparation and delivery of L P D nanoparticles Move Preparation and delivery of L P D nanoparticles MATERIALS reagents 5 X glucose in water (26 % m/V), sterile Dissolve 13 g of glucose in about 40 ml of sterilized water, then bring the volume to 50 ml with sterilized water, filter through a 0-2 Mm filter and store at room temperature for up to one year. DOTAP (25m g/m l of storage solution), dissolved in chloroform (AvantiPolarLipids). DOTAP (1, 2-dioleoyl-3-trimethylaminopropane) is one of the most widely used cationic lipid materials for gene delivery. Some electroneutral lipid materials (e.g., DOPE, 1,2-dioleoyl phosphatidylethanolamine, cholesterol) can be used to improve the efficiency of transgenesis. Cholesterol in chloroform solution (20m g/m l) (Sigma-Aldrich). When used in vivo, cholesterol can be used as a co-lipid in place of DOPE to produce more stable and effective cationic liposomes (LietaI. 1998). Plasmid DNA dissolved in deionized water (l m g /m l) Plasmid DNA should be highly purified and free of endotoxin. Water or 5.2 % glucose solution is recommended. Dilution of DNA with 5.2 % glucose solution is recommended because the salt interferes with the interaction of DNA with cationic liposomes and agglomerates. Ichthyocyanine sulfate (100 m g/m l, Ekins-Sinn) Ichthyosperm sulfate (MW 4000-4250), an arginine-rich cationic peptide, is used in LPD to interact with DNA. Imol sulfated ichthyoglobulin contains 21 mol of positive charge and interacts strongly with DNA molecules. Bacteriostatic water Steam sterilization at 121°C for 30 min. Apparatus Ultrasonic water bath Corex glass tubes (30 ml) LiposoFast extruder with IML injector (Avestin) Nitrogen (N2) cylinder Polycarbonate membrane filters (l. 0 M m, 0.4 f x m, and 0 - l) n m, C o r n i n g Corporation) Polyethylene centrifuge tubes (50 m l ) Vacuum dryer Methods Preparation of cationic liposomes 1. Rinse the 30 m l C o r e x glass test tube 3 times with chloroform. The residual chloroform can be left dry 2 . In a glass tube mix ○ - 8m l of D O T A P reservoir solution with ○.55m l of cholesterol reservoir solution. Currently, cationic liposomes are prepared in our laboratory by mixing D O T A P and cholesterol at a molar ratio of I : 1 . The charge ratio of Iip0pIex prepared from 1 hong of plasmid D N A and cationic liposomes containing 3 6 _ 1 D O T A P and 36_1 cholesterol is 12 : 1 ( + : -). 3- In a chemical fume hood, with the glass tube rotated, nitrogen is introduced along the wall of the tube, causing the chloroform to evaporate and a film to form at the bottom of the tube. The process can be accelerated by placing the glass tube in a 35°C water bath. 4 . Place the glass tube in a vacuum desiccator for 2~3 h to dry completely. To prevent the loss of lipid film under vacuum, cover the glass tube with aluminum foil and at the same time prick some small holes in the foil with a needle. 5. Add 2m l of sterilized water into the glass tube. 6 . Vortex at maximum speed for 15s to resuspend the lipids until no lipids remain on the walls of the tube and there is no white precipitate. For resuspension, the glass tube can be sonicated intermittently in an ultrasonic water bath for several times (100~l5s). 7- Leave the resuspension solution at room temperature for 2~3 h or incubate overnight at 4°C to fully hydrate the lipids. The longer the hydration time, the easier the liposomes can be extruded. Overnight hydration is recommended. 8- Vortex the suspension and incubate in a water bath at 65°C for 5 to l o m i n. If lipid aggregation is observed, sonicate the resuspension in an ultrasonic bath until the aggregation disappears, and then place it back in the water bath. 9. Place two I.O p m polycarbonate films in the extruder. 10-Heat the extruder to 65° C for 5 m i n . Heating of the lipid suspension and the extruder to 65°C is necessary to maintain the lipid liquid state, to facilitate extrusion and to improve the efficiency of lipid blending. 11. Extrude the suspension through the filter membrane and repeat 5 times. Return the extruder to the water bath. After extrusion, the suspension changes from cloudy to translucent. 12- Repeat steps 9 to 11 consecutively using 0. 4um and 0.1um polycarbonate filter membranes to obtain liposomes with an average particle size of 1 ○○ ~ 200n m. The liposomes were then extracted into a water bath. The cationic liposome suspension can be stabilized at 4°C for several months. I i p o p l e x preparation 13- Add 60ul of 5 X glucose solution, 154ul of sterilized water and 86ul of cationic liposomes to a 50m l centrifuge tube and vortex at medium speed for 10s. Liposomes and plasmid DNA are mixed under mild conditions to obtain the lipid complex. A final volume of 600ul of solution contains 100ug of plasmid DNA. volume and mass are increased proportionally when larger quantities are required. 14. Add 40 ul of l5X glucose solution, 60 ul of sterile water and 100 ul of plasmid DNA to a 1.5 ml centrifuge tube and tap the tube to mix. Do not vortex. 15. While gently vortexing the diluted liposome suspension, add the diluted DNA solution dropwise. Transfer the liquid with the tip of a 10-ul pipette; it takes 5 to 10 s to add 200 ul of DNA solution. 16. Prior to injection, incubate the complex at room temperature for 10 to 15 m i n . Preparation of LPD 17- Add 60u; 1 5 X glucose solution, 148ul of sterilized water and 86ul of cationic liposomes, and 6ul of ichthyoglobin sulfate solution in a 50m l centrifuge tube and vortex for l0 s at medium speed. The optimal composition of the LPD nanoparticles was 12n m ol of DOTAP/12n m ol of cholesterol/0- 6ug of caviar sulfate/I ug of plasmid DNA, _ The charge ratio of DO TA P and D N A was 4 : 1, and the charge ratio of caviar and D N A was 1 : 1. 18- Add 40M 15 X glucose solution, 60ul of sterile water and IOOfJ plasmid DNA to a 1.5 ml centrifuge tube and tap the tube to mix. Do not vortex. 19- While gently vortexing the diluted liposome-cisplatin solution, slowly add the diluted D N A solution drop by drop. Transfer the liquid with the tip of an IOOOul pipette; it takes 5-10s to add 200ul of DNA solution. L PD nanoparticles are usually prepared immediately before use. However, LPD can be stored at 4°C for at least 4 weeks without loss of viability. LPD can also be lyophilized as a powder and stored at room temperature for at least one year without loss of viability upon resuspension (Lie ta L 2000). 20 - LPD is incubated at room temperature for 10 to 15 m i n and then injected into the appropriate site of the animal. 2 1- Detect the expression of D N A by a suitable method. For more product details, please visit Aladdin Scientific website.