Experiments with biodegradable nanoparticles
Experiments with biodegradable nanoparticles
Biodegradable nanoparticles are colloidal particles, typically around IOOnm in diameter, which can be prepared from biodegradable polymers such as polylactic acid-polyglycolic acid (PLGA) or polylactic acid. These nano-particles enter the cell by endocytosis and have been shown to rapidly escape from the lysosome, thus protecting the nano-particles and the DNA contained therein from degradation in the lysosome (Panyam etal. 2002). Therefore, plasmid DNA contained in nanoparticles is protected from degradation by extracellular and intracellular nucleases. The slow release of DNA from the nano-particle allows for sustained gene delivery and prolonged gene expression, thus the biodegradable nano-particle, unlike other non-toxic gene delivery systems, is a carrier of sustained gene expression.
Author: T. Friedman et al, Translator: Jingwei et al. This experiment is from "Gene Transfer".
Operation method
Preparation of biodegradable nanoparticles and their application in gene transfection Move Preparation of biodegradable nanoparticles and their application in gene transfection MATERIALS reagents Acetylated bovine serum albumin (Ac-BSA; Sigma-Aldrich) Cell Lysis Reagent (CCLR; Promega) or Triton X-100 (1.0 ¾ m/V) solution. Triton X-100 (1.0 ¾ m/V) solution Cells to be Transfected Chloroform Fetal Bovine Serum (FBS) Water (sterile) Isopropyl alcohol Fluorokinase substrate (if fluorokinase is used as a marker gene) (Promega) Cell culture base Complete culture base (RPMI 1640 or other culture fluids depending on cell line) containing 10 % (m/V) fetal bovine serum RPMI 1640 or other serum-free culture fluids (depending on cell line) BCA protein test (Microro BCA kit, Pierce) Phosphate Buffer (PBS, p H 7.4) Plasmid DNA (10m g /m l ) Poly(lactic acid)-poly(hydroxyglycolic acid) copolymer (PLGA, poly(lactic acid)/poly(hydroxyglycolic acid) = 50:50, specific viscosity in hexafluoroisopropanol I.32dl/g; Birmingham Polymers Inc. Polyvinyl alcohol (PVA) (Molecular mass 30,000-70,000; 87%-89% hydrolysis) Tris-EDTA Buffer (pH 8.0, sterilized) 0, sterilized) Instrumentation Centrifuge (50 ml) Lyophilizer Membrane (〇 -22um; Millipore) Glass tube (5mL) Ice bath 37°C incubator Microcentrifuge Centrifuge Tubes 2 4-well plates Constant temperature oscillation bath (37°C) Ultrasonic processor (X L 2015 sonicator ultrasonic processor ; Misonix Inc.) Ultraviolet spectrophotometer Vacuum drying oven Methods Nanoparticles containing plasmid DNA are produced by emulsified solvent volatilization and can be prepared with 30 to 90 mg of polymer per run. The method with 30 mg of polymer is described below. Preparation of solution 1. Preparation of polymer solution Preparation of polymer solution: 30 mg of PLGA was dissolved in I ml of chloroform in a 5 ml glass tube with stirring, and it took 3-4 h to completely dissolve the polymer. 2 . Preparation of plasmid DNA solution: Mix I mg of plasmid DNA solution (l0 mg/ml) with EOOfXlTris-Edta buffer, add 2 mg of acetylated bovine serum protein without nuclease without vortexing, and tap the test tube to dissolve the BSA. It takes approximately 3 h to completely dissolve the BSA. Alternatively, the DNA solution can be stored overnight in the refrigerator with the BSA to completely dissolve the BSA. 3 . Preparation 2. Prepare 2.0 % (m/V) PVA solution: Slowly add 0.2 g of PVA (30 to 70 molecular mass) to an omega-cooled Tris-Edta buffer under stirring, requiring approximately 30 mIn to dissolve the PVA. Centrifuge at 4°C, lOOr/min, for 10 millimeters and filter through a 0.22-mm sterile membrane to remove the insoluble PVA. Saturate the PVA solution with IOul of chloroform. DNA encapsulation 4 . Add the above DNA solution to two 100-ul portions of polymer solution and vortex for a few millimeters to form a water-in-oil emulsion. 5 . The emulsion was sonicated for 2 min in an ice water bath using a probe sonicator with an output power of 55W. This process reduces the droplet size of the emulsion. The ultrasonic probe was cleaned with isopropyl alcohol prior to sonication, and the probe was placed in the center of the emulsion and prevented from contacting the wall of the tube during sonication. 6- Add the emulsion in two portions to a 50 ml centrifuge tube containing 6 ml of PVA and vortex for a few millimeters after each drop to form a water-in-oil-in-water (water-in-oil-in-water) double emulsion and sonicate for 5 millimeters at step 5. The emulsion is slowly stirred overnight (about 18 h) in the same tube at room temperature to allow the chloroform to evaporate. 8. To ensure complete volatilization of the chloroform, the suspended nanoparticles were stirred in a vacuum dryer and dried for another hour. 9. Collect the nanoparticles by ultracentrifugation at 4°C, 110 OOO g for 20 m i n and collect the supernatant at the same time. 1 0 . Resuspend the nanoparticles in 5 ml of Tris-Edta buffer. The nanoparticles were first washed repeatedly with the buffer until they were fully suspended, and then ultrasonicated for 30 s in an ice bath at step 5. 11. Repeat steps 9 and 10. Repeat steps 9 and 10 to remove free DNA and PVA. 12- Determine the amount of unencapsulated DNA using the supernatant from Step 9 and the washing solution from Step 11, and measure the absorption at 260 nm using an ultraviolet spectrophotometer. The supernatant from Step 9 and the washing solution from Step 11 for the preparation of DNA-free nanoparticles were used as reference in the test. 13 - Resuspend the nanoparticles in 1 to 2 ml of sterile water and sonicate for a period of lmillion. Centrifuge the nanoparticle suspension at lOOOr/million at 4°C to remove possible nanoparticle aggregates. 14. Collect the suspension and store in weighed centrifuge tubes (in several portions if necessary) and freeze at 80 °C for 45 m i n. Freeze-dry for 2 d to obtain a dry solid powder. Store the freeze-dried nanoparticles at 4°C. Transfection methods 15. In 24-well plates, inoculate the cells to be transfected at a density of 3.5X 104 cells/ ( well . m l ). Cells to be transfected are inoculated in 24-well plates at a density of 3.5X 104 cells/(well . ml) in 6 parallel wells using complete medium. The same cell density was used without the Ghanaian rice pellet as a control. 16. Cells were cultured in 24-well plates for 24 h. The cells were then incubated in a 24-well plate. 17. Resuspend 4mg of nanoparticles loaded with DNA in a sterile environment with 0.5 ml of RPMI 1640 (other serum-free cultures may also be used for different cells). Sonicate for l 0 m in in a water bath sonicator. 1 8 . Dilute the nanoparticle suspension to 9 m l with R P M I 1640 containing 10 % F B S (or other nutrient solution suitable for cell growth). 19. Aspirate the nutrient solution from the cell culture plate and add X l m I of the above nanoparticle suspension. 20- Incubate the cells for 24 h at 37°C, 5 % C 02 . 21. Aspirate off the nanoparticle suspension and add an equal amount of cell culture medium. Subsequently, the culture medium was changed every other day without the addition of nanoparticles. 22.The cells were lysed at the appropriate time to measure the level of gene expression. a. Rinse the cells twice with cold P B S. b. Add 0 - 1m l I X Cell Lysis Solution (C C L R ) or 0 - 1 % U/V ) Triton X -100 solution to each well. c. Shake the 24-well plate at 37 C for 3 0 m i n in a shaking incubator. 23. Detect the level of gene expression in the cell lysate. If dongleuclease is used as a marker gene, the gene expression level can be detected by chemiluminescence using the fluorophore substrate, and the protein content can be determined using the B C A protein quantification reagent to normalize the expression level of shikleuclease per milligram of cellular protein. For more product details, please visit Aladdin Scientific website.