Experiments on nanogene vectors based on polylactic acid and polyethylene glycol
Experiments on nanogene vectors based on polylactic acid and polyethylene glycol
Nanoparticles combining polylactic acid-hydroxyglycolic acid (PLGA) and polyethylene glycol (PEG) derivatives have been used as gene carriers for mucosal permeation. The advantages of these nanocarriers are due to their inherent biodegradability, low toxicity of the components, and mild preparation conditions. Additionally, for in vivo application, the following advantages are noted: (1) adequate DNA loading rates; (2) the ability to control DNA release while preserving DNA conformation and bioactivity; and (3) the ability to overcome nasal mucosal barriers to DNA vaccine delivery to cells, which induces a more robust systemic immune response compared to coded proteins. Author: T. Friedman et al, Translator: Jingwei et al, This experiment is from "Gene Transfer".
Operation method
Preparation of P L G A and P E G based nanogene vectors Move Preparation of P L G A and P E G based nanogene vectors Materials reagents Agarose gel (1 %) Ethanol Ethyl acetate Methylene dichloride Phosphotungstic acid coloring solution (2 %) Picogreen reagent (Molecular Probes, Invitrogen) Plasmid: pCMV-(3-gal carrier (ElimBiopharmaceuticals) Poloxamer: with different hydrophilic-lipophilic ratio (H L B) and different chain lengths Piuronic F 68 and Piuronic L 121 (BASF, Germany). Poloxamine: with different hydrophilic-lipophilic ratios (H L B) and different chain lengths Tetronic 904 -¾ Tetronic 908 (BASCOM, Belgium) Polylactic acid-polyethylene glycol block copolymers (PLA-PEG, Alkermes, Ohio) Polylactic acid-polylight-glycolic acid copolymers (PLGA, Boehringer Ingelheim) Polyvinyl Alcohol (PVA) solution (1 % and 0.3 % m/V) (1 % and 0.3 % m/V) ELISA reagent Instrumentation Agarose gel electroswimming equipment and enzyme labeling for ELSA Ultrasonic Wave Breaker (Sonifier, Branson, Barcelona, Spain) Fluorescence spectrophotometer Transmission electron microscope Vacuum dryer Particle Size - Surface Potentiometry Zetasizer 3000 H S (Malvern Instruments) Methods Preparation of nanoparticles 1 . Preparation of nanoparticles with a specific composition Hybrid PLGA/PEO derivatives Nanoparticles Prepared by solvent displacement technology. a- 500 Kilograms of plasmid DNA was dissolved in 200ul of aqueous phase. b. Dissolve 50mg of PLGA and 50mg of PEO derivative in 2ml of methylene chloride. c. Mix the water phase from step a with the oil phase from step b and vortex to form a water-in-oil emulsion. d. Add 25 ml of a polar solvent (e.g., ethanol) to the emulsion to precipitate the polymer in the form of nanoparticles. e. Add 25 ml of water and allow the organic solvents to fully evaporate. Collect the nanoparticle suspension. PLA-PEG Nano Particles They can be prepared using the solvent displacement technology described above or by double emulsion technology. a- Dissolve 200 ug of plasmid DNA in 2(%1) aqueous solution. b. Dissolve 40 m g of plasmid-PEG in I.5 ml of ethyl acetate:methylene chloride (I:1) solvent. c. Mix the aqueous phase from Step a with the oil phase from Step b. Ultrasonicate for 5 s. d- Add the above emulsion to the aqueous solution of PVA (l %; n/V) and sonicate for 5s (output power = 10) to form a double emulsion (oil-in-water - water-in-oil). e. Add the above emulsion to 50 ml of 0.3% (m/V) solution under solicitation. e. Add the above emulsion to 50 ml of 0.3 % (m/V) PVA in water under stirring gently to solidify the nanoparticles. f. Evaporation in vacuum to remove organic solvents. Separation of nanoparticles 2, 15°C , centrifugation at a speed of 10 ○○ g allowed the separation of nanoparticles. Such conditions allow for maximum precipitation of nanoparticles without precipitating free plasmid DNA. 3- Resuspend the nanoparticles in water and gently vibrate or vortex. Characterization of rice grains 4- Measurement of the grain size of nano grains by grain size analyzer (Zetasizer H S 3000). 5- Measurement of the potential and distribution with the Zetasizer H S 3000. 6. The size and morphology of the nanoparticles were visualized with a transmission electron microscope by dyeing with phosphotungstic acid (2%). 7- Fluorescent reagent was used and measured by fluorescent photometer according to the procedure provided by the reagent supplier. The binding efficiency of DNA was measured using a fluorescence photometer according to the procedure provided by the reagent supplier (Molecular Probes, Invitrogen). 8 . Analyze the structural integrity of released plasmid DNA or plasmid DNA extracted from nanoparticles by 1 % agarose gel electrophoresis. In vivo gene expression In vivo gene expression can be studied by examining the immune response of mice to coded proteins. 9- Drops of IOfJ-suspended nanoparticles (total 2.5ugC M V-p-gal, 3 drops per nasal cavity) were administered to the nasal cavity of mice (6-9) at 15million intervals. 10. Blood is collected from the tail of the mouse by vein sampling at a predetermined time, centrifuged at 3000 g for 5 m i n at 4°C, and the serum is collected and stored at 20°C. The blood is collected at the same time as the blood from the mouse. The ELISA method is used to determine the level of Ig G and Ig A. The ELISA method is used to determine the level of Ig G and Ig A in the blood. For more product details, please visit Aladdin Scientific website.