PEI Nanoparticles for Targeted Gene Delivery
PEI Nanoparticles for Targeted Gene Delivery
P E I Due to its polycationic nature, it and negatively charged D N A molecules form nanoparticles through electrostatic interactions, which further help D N A to pass through extracellular and intracellular barriers. Recently, the development of receptor-mediated endocytosis, intron escape, and nuclear raking to transgene carrier materials has attracted extensive attention, typically by coupling functional molecules to cationic polymers via chemical bonds to achieve selective delivery of D N A to target cells. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Preparation and transfection of PEI/DNA nanoparticles Move makings reagents Dimethyl sulfoxide (D M S O ; Sigma-Aldrich) D M E M (Dulbecco's modified Eagles' m e d i u m ) cell culture medium with 10 % fetal bovine serum (F B S ) Lithium chloride (LiCl, Sigma-Aldrich) Fluoresceinase assay reagent (P r o mega) Mammalian cells (exponential growth phase) Serum-free Opti-M E M Cell Culture Medium (Invitrogen) PEI polymers (M.W. 600 ~ IOOOkDa, Fluka; M.W. 750kDa, 25kDa, 2kDa and 800Da, Sigma-Aldrich; M.W. I. 2kDa, IOkDa or 70kDa, Polysciences) Peptides, prepared by solid-phase chemical synthesis method Phosphate buffer (P B S ) 0 -lmol/L sodium phosphate buffer solution (p H 7. 4) 0.15m o l/L NaCl Plasmid DNA, coding for fluorophore reporter gene 5X reporter gene lysis buffer (P r o mega), diluted to 1x with P B S Ultrapure water Instrumentation D c Protein Assay Kit (Bio-R a d ) Dialysis Membrane lyophilizer Fluoresceinase Tester Magnetic stirrers and magnets Reaction bottles Tissue culture flasks Methods Activation of PEI with crosslinker 1- Prepare SMC storage solution (50 m m o l /L, dissolved in DMSO) 2 . Prepare a 10mg/ml solution of PEI with DMSO. Add 2~5mg LiCl to increase the solubility of PI. 3 - Slowly add SMC to the solution using an injector and react for 2 hours at room temperature. The dosage of SMCC solution is calculated on the basis of the desired molar ratio of SMCC to P EI. 4 - The modified PEI is analyzed in ultrapure water for 2d with at least 5 water changes per day. 5. Collect the solution from the analyzer bag and freeze-dry the sample. Coupling of activated PEI and peptides 6 . Dissolve the peptide with P B S as a solvent and prepare a 20~50m m ○ l/L solution. 7 - Dissolve activated P E I (step 5) with P B S as solvent and prepare a solution of l O m g /m l . 8- Slowly add the peptide solution to the activated PEI and react for 24 h at room temperature. The amount of peptide depends on the molar ratio of the final desired coupling. 9. Dialyze the peptide-coupled P E I in ultrapure water for 2d with at least 5 water changes per day. 10. Collect the solution in the dialysis bag and lyophilize the sample. Preparation of P E I / D N A complexes 11. Preparation of storage solutions a. Prepare a storage solution of l m g /m l of plasmid DNA in ultrapure water. b . Configure the storage solution for P E I (step 5 ), or the storage solution for peptide-coupled p EI (step 10 ), with l ○ n m m ○ l/m l of amino N (p H 7. 2). c-Preparation of the ternary complex requires the preparation of a 5 mg/ml stock solution of the targeting peptide containing the D N A binding sequence in ultrapure water. Steps 11 to 22 require preparation of the complexes in a sterile environment using sterilized reagents at room temperature in a flat-flow ultraclean bench. 12-Preparation of working solution a- Dilute Iug of plasmid D N A into 50ul of serum-free Opti-M E M (for transfection of cells in 24-well plates). b. With 50ul of serum-free Opti-M E M , dilute P E I containing the appropriate amount of P E I or peptide-coupled P E I . c. Prepare the ternary complex by diluting the appropriate amount of peptide with 50ul of serum-free Opti-MEM. 13. Add the peptide-coupled P E I dropwise to the D N A solution under worm-rotation oscillation. For ternary complexes, the peptide is added dropwise to the DNA solution under worm-rotation oscillation. 14. Incubate at room temperature for 30 m i n . Peptide-coupled PEI/DNA complexes can also be used directly for transfection (step 16). 15. To prepare the ternary complex, add the PEI solution dropwise to the target peptide/DNA complex solution under worm-rotation shaking and incubate for 30 m i n at room temperature. The ternary complex can also be used directly for transfection (step 16). Transfection Assay 16-The day before transfection, mammalian cells were inoculated into 24-well plates at a density of 50 O O O O per well and incubated for 24h at 37°C , 5 % C O 2 using D M E M complete medium containing 10% fetal bovine serum. 17 The medium was aspirated and the cells were washed 2 times with PBS (preheated at 37°C). 18-Perform the following operations in order: Add 100~150ul of serum-free Opti-M E M to each well. Add 100~15 〇 4 D N A complex containing 1 plasmid D N A per well (Step 14 or 15). Incubate the cells at 37℃ with 5% CO2 for 4 hours. 19- Aspirate the transfection solution. Wash with PBS (preheated at 37°C) for 2 times. Add I ml of DMEM complete medium containing 10 % fetal bovine serum and incubate the cells for 24 to 48 hours. 20. Detect the expression of fluoresceinase. Add IOOul of I X reporter gene lysis buffer to each well and lysed the cells. 21- To detect the activity of fluorokinase, 20ul of cell lysate was added to IOO ul of fluorokinase assay reagent and fluorokinase activity was detected by fluorokinase assay. 22- To normalize the fluorokinase activity, a D . Protein Assay Kit to determine the protein content of the cell lysate. For more product details, please visit Aladdin Scientific website.
S M C C [succinimidyl-4-(N -maleimidomethyl)cyclohexane-l-carboxylate] (Pierce)
SMCC is easily hydrolyzed. Configure the stock solution with anhydrous DMSO under dry H protection. The storage solution is stable for 3 months at 4°C. Steps 1 through 10 are to be performed in a chemical fume hood, following chemical safety practices. "