Solid lipid nanoparticles (S L N ) have many technical advantages over gene carriers such as cationic liposomes and cationic polymers. However, in the absence of lysosomal reagents (e.g., chloroquine), even if the components of the S L N vectors are optimized, their gene transfection efficiency is still lower than that using standard transfection reagents. In this paper, we increased the gene transfection efficiency by doping S L N with the H I V -I protein dimer T A T polypeptide and showed that S L N doped with T A T had a higher transfection efficiency and reduced toxicity than standard transfection reagents. Author: T. Friedman et al, Translated by W. Jing et al. This experiment is from "Gene Transfer".
Operation method
Preparation of the in vitro transgenic vector S L N Move Preparation of the in vitro transgenic vector S L N Materials reagents Cell line to be transfected serum-free cell culture base for remote cell lines Fetal Cow Serum (F CS) Gentamicin (Invitrogen 15710-049) H B S (HEPES Buffered Biological Salt Water) 150 m m o l / L N a C l 10 m m o l / L H E P E S ( p H 7. 4) Penicillin/streptomycin (G I B C O 15140-122) Plasmid D N A Cetyl palmitate (Henkel, Diisseldorf, Germany) D O T A P (Sigm a-Aldrich) S p a n 85 (ICI Surfacants, Everberg, Belgium) T w e e n -80 (ICI Surfacants, Everberg, Belgium) SLN is prepared by hot high-pressure homogenization (Muller etal. 2000b). The solid lipid is heated to l0°C above its melting point. The surfactants Tween-80 and Span 85 are mixed 7:3. The mixture (2 % m/m) was mixed with 1 % (ot/ot) of the cationic lipid DOTAP in hot aqueous solution, further mixed with the basic lipid cetyl palmitate (4 % m/m), stirred at high speed for 1 min to form a pre-emulsion, which was then homogenized four times with a high pressure homogenizer at 85°C and 480 bar. TAT2 peptide The sequence of TAT 2 peptide is C (Y g r k k r r q r r r r g )2, which is rich in arginine. The TAT2 peptide was synthesized by an automated peptide synthesizer according to the standard FM0c method, purified by reversed-phase high-performance liquid chromatography (HPLC) and analyzed by mass spectrometry. The free sulfhydryl group was modified by a disulfide-dipyridine reaction (Plank etal. 1999). This modification is important to avoid the formation of disulfide bonds in solution for the T A T 2 polypeptide. Instrumentation. Automatic synthesizer (431A; AppliedBiosystems) Cell culture plates (2 4-well) High-pressure homogenizer (EmulsiFlex-B 3; Avesti n l n c , O t t a w a , C a n a d a ) High Speed® Mixer (Ultra Tourrax T 25; Jahnke and Kunkel, Gremany) Incubator (37°C, 5 % CO2) Reversed-phase high-performance liquid chromatography (HPLC) Methods Synthesis of SLN-gene vector complexes 1 . Prepare the following solution for each well of a 24-well plate. a . Dilute l ug of plasmid D N A with H B S to 50 ul. b . b - Dilute 0. 65 ug of TA T2 peptide with H B S to 50 ul . c - Dilute 2. 5 tonsug SLN to 50m1 with HBS. 2. To prepare T A T 2-D N A complex, add plasmid D N A solution to T A T 2 solution. Blow out 10 times by pipetting to mix well, and incubate lO m in at room temperature. 3- To prepare the ternary complex, add the S L N solution to the T A T 2-D N A complex. The solution was blown out by pipetting for 1 time to mix thoroughly and incubated for lO min at room temperature. In vitro transcription experiments 4-The day before transfection, cells were inoculated in 24-well plates with medium containing F C S . 5- Aspirate the cell culture medium from each well and replace it with 850^1 serum-free medium. 6 . Add 150ul of S L N ^ Gene Vector Ternary Complex Solution (made in step 3) to the cells. Incubate at 37°C for 4 h at 5 % C O 2 . 7- Aspirate the transfection solution. Continue the incubation with medium containing 10% FCS, 0 . 1 % penicillin/streptomycin and 0.5% (V/V ) gentamicin. 8- The efficiency of gene transfection was tested at the indicated time. For more product details, please visit Aladdin Scientific website.
Solid Lipid Nano Particles (SLN)
Cells should have a cell density of 60 % to 70 % at the time of transfection.