This chapter describes the detailed experimental procedure for encapsulation of nucleic acids by nanolipid particles (N L P ). This N L P carries a polyethylene glycol (P E G ) shield and contains biosensitive lipids and ligands for targeted delivery of D N A . Unlike conventional cationic lipid-D N A complexes (Iipoplex), the N L P described above was obtained by ethanol dialysis and was doped with P E G-conjugated lipids in a lipid bilayer surrounding the D N A molecule, with particle diameters of less than l00 n m . Author: T. Friedman et al, Translated by W. Jing et al. This experiment is from "Gene Transfer".
Operation method
Preparation of natriuretic granules Move Preparation of natriuretic granules Materials reagents Cationic Lipids (5m g /m l ) Cationic lipids can be either reductant-sensitive lipids, such as TC L, or reductant-insensitive lipids, such as 1,2-dioleoyl-3-trimethylaminopropane (D O T A P ) (Fig. ID ). T CL lipids: (2-aminoethyl)-dimethyl-(2-[3-cholesterol disulfide ethylammonium] ) ammonium bromide hydrochloride was synthesized by reference to a literature method (H u a n g etaL 2005) and dissolved in methanol/chloroform (1 : 1, VAO) to prepare a 5 m g / m l storage solution. DOTAP (AvantiPolarLipids [Alabaster, Alabama]) was dissolved in chloroform to prepare a 5 mg/m l stock solution. Chloroform Ethanol Cofat (100 m g/m l) The membrane fusion lipid 1, 2-dioleoylglycerol-3-phosphoethanolamine (DOPE) (Figure 1E) is a widely used auxiliary lipid that promotes the efficiency of gene transfection. DOPE (Avanti Polar Lipids) has a relatively small hydrophilic head and is believed to induce cell membrane fusion, thus helping DNA to escape from introns into the cytoplasm during gene delivery. Ligand - P E G Lipid (0.05m m o l/L ) The sequence of the T A T polypeptide is CGGGRKKKRRQRRRGYG. It can be attached to maleimide-functionalized PEG lipids via an N-terminal cysteine residue: 1, 2-distearoylglycerol-3-phosphoethanolamine ^N-maleimide (polyethyleneglycol) 2000 (AvantiPolarLipids) to prepare "TAT-PEG■ lipids". To learn more about the chemistry, see reference Hermanson, (1996). "T/ff-PEO lipids can be dissolved in methanol/chloroform (I:I, V/V) solution to prepare a 0. 5 1/L storage solution. 0. 5 1/L storage solution. Other ligand-PEG lipids can be used instead of TAT-PEG-lipids. Mammalian cells (in exponential growth phase) Mouse (female, 6 to 8 weeks old) Methanol PEGylated lipid (10m g /m l) PEGylated lipids can be either low pH-sensitive lipids, such as PODS (Figure 1A), or pH-insensitive lipids, such as PEOL, 2-distearoyl-"r glycerol-3-phosphonoethanolamine (PEODSPE) (Figure IB). The synthesis of PO D S can be referred to the literature Guo et al. (2004). P E O D S P E can be purchased from Avanti Polar Lipids. The PEGylated lipids have a molecular mass of 2000 PEG chain segments and can be dissolved in chloroform at a concentration of lOmg/m l. The molecular mass of the PEGylated lipids is 2000. Plasmid DNA (Advysys or other gene vector manufacturers) Plasmid DNA is purified in the laboratory, using commercial DNA purification kits. s i R N A or oligonucleotides (Invitrogen or other oligonucleotide manufacturers) I O X T r i s Buffer (50 m m o l /L , p H 7. 4 or 8. 5) The pH 7.4 buffer is used to prepare N LPs containing PEG lipids that are not sensitive to PH, while the pH 8.5 buffer is used to prepare N LPs containing PEG lipids with low PH sensitivity (PODS). dilute IOXTris Buffer to IX Buffer prior to use. Reserve 5 ml of IOXTris Buffer (filtered through a 0.22" pore size membrane) to suspend the lipids and dissolve DNA. IXTris Buffer Biological Saline (TBS) (p H 7.4 or 8.5) 50 m m o l / L Tris I. 5 m o l / L N a C l Adjust p H to 7.4 or 8.5. p H 7.4 buffer is used to prepare NLPs containing P EG lipids that are not sensitive to p H, and pH 8.5 buffer is used to prepare NLPs containing P EG lipids with low PH sensitivity (PODS). dilute to pH 8.5 before use. Instrumentation Analyzer (Pierce) The dialysis unit consists of a Slide^A-Lyzer dialysis tubing with a 10,000,000 molecular mass cutoff and a floating cap. Constant temperature water bath (55°C ) Rotary Evaporator (Brinkmann Instirumenl:) The lipid drying unit is used to remove large amounts of solvent from lipid mixtures and to deposit lipid films on glass surfaces. Lipid films were deposited into vacuum-resistant glass round-bottom flasks (300 ml) and dried for at least 3 h to remove traces of organic solvents from the films. Sterile injector (3 ml) and needle (18 G I.5) (Beckon Dickinson) Methods Ethanol dialysis method for encapsulating nucleic acids into N L P 1. Aspirate PEGylated lipids, cationic lipids, and DOPE from the storage solution, mix thoroughly in a 16 mm X IOO m m glass tube with a screw cap (cleaned and sterilized prior to use), and dry under reduced pressure using a rotary evaporator to remove the solvent. The glass tube containing the lipid film is then dried under higher vacuum for at least 3 h, preferably overnight. Typical preparations contain 20 umol of total lipid, where the lipid molar ratio is: PEGylated lipid: cationic lipid: co-lipid = 10 : 50 : 40 or 10 : 30 : 60. Use a glass pipette to measure and transfer lipids dissolved in organic solvents. The lipid storage solution must be sealed with PTFE tape immediately after each operation and stored at 120°C. If the sample contains both the cationic lipids and the co-lipids, it should be stored in the laboratory. If both TCL and PODS are present in the sample, to avoid degradation of PODS during drying, morpholine (20:1 molar ratio to TCL) should be added to the lipid mixture prior to the addition of PODS. 2. Dissolve the dried lipid in anhydrous ethanol for 30 m i n , then add 0.5 m l of I X Tris buffer by pipette, and vortex for 2 m i n at maximum speed rapidly to form a single-phase solution of lipid (solution A). To accelerate the dispersion of lipids in ethanol, the lipid mixture containing TCL can be heated under argon protection for 0.5 min at ~65°C before mixing with the Tris buffer, then cooled naturally at room temperature for 5 min. It is not uncommon for some of the lipids to precipitate in cold ethanol. 3. Solubilize I.I O O m g of nucleic acid (plasmid DNA or other oligonucleotides) in a mixture of 0 -5 m l of I X Tris buffer and 0. 5 m l of ethanol (Solution B ). During sample preparation, tighten the caps of Solution A and Solution B to minimize ethanol evaporation. 4. Typically, DNA solution (Solution B) is injected into the lipid solution (Solution A) over I s using a 3 ml syringe with an 18 GL 5 needle. To fully emulsify the solution, the syringe is used to aspirate and blow the mixture 10 times over 15 s. The mixture is vortexed at maximum speed for 30 s. The solution is then vortexed at maximum speed. 5. Use a 3 ml syringe with an 18 G 1.5 needle to transfer the lipid-DNA mixture into the Slide A-Lyzier dialysis tubing. Dialyze for 2d at 4°C with I L of IX Tris buffer, changing the dialysis buffer 4 times during this period. Submerge the tubing in the dialysis buffer and allow it to hydrate for 30 s before use. Be careful not to touch the dialysis membrane with the needle during the transfer of the solution. 6. Replace the dialysis buffer with IX TBS buffer instead of IX Tris buffer and continue dialysis overnight at 4°C. IX TBS buffer contains 150 mmol/L NaCl, which is more suitable for in vivo dialysis than the lower ionic strength IX Tris buffer. 7- Aspirate the N L P sample from the dialysis tubing using a 3 m l syringe with an 18 G 1. 5 needle, transfer to sterilized tubing with a screw cap, and store at 4°C . Modification of NLP surfaces by disulfide bond exchange reactions 8- Reduction reagents are ready to use. Prepare a 0.1 to 0.2 mOl/L solution of cysteine in pure water or IXTBS buffer with a pH of 8.5 (for samples containing PDS). 9- Add the reducing reagent to the NLP solution (step 7) at a molar ratio of 10:1 (10 times the amount of TCL in NLP). Vortex for 10 s and leave at room temperature for 5 m i n , then transfer the sample to a dialysis tube and rapidly analyze for I d with I L I X T B S buffer at 4°C to remove excess reducing reagent, changing the dialysis buffer once. Insert "ligand-PEG-lipid" on the N LP surface 10- Determine the amount of TAT Peptide-PEG-Lipid, which is equal to 0-3 % of the total amount of N LP Lipid. Transfer the T A T peptide-P E G -lipid storage solution containing the above amount to a 16 m m X 10 ○ m m sterilized glass tube with a screw cap. Remove the organic solvent by rotary evaporation under reduced pressure and dry overnight under high vacuum. 11- Hydrate the T A T - P E O D P P E lipid film directly with N L P solution at room temperature I O m i n , then incubate in a 55°C water bath. Be sure to charge with argon or nitrogen before sealing the nut in operation. Remove the glass tubes and vortex for 10 s every I O m i n during incubation. These treatments cause the transfer of TAT-PEG lipids from the glass surface into the N L P by micelle conversion. Refer to steps 12 to 13 for in vitro transfection experiments; for in vivo transfection experiments, refer to steps 14 to 15. In vitro gene transfection 12. Prior to performing in vitro transfection experiments, mammalian cells, such as C V -I cells, are cultured overnight in 24-well plates with complete medium containing 1 0 % fetal bovine serum, grown at a density of I X l O 5 cells per well. 13-Change the medium by adding N L P encapsulated with ZfjtgD N A to the wells and incubate for 4 to 48 h (during which time the medium may or may not be changed). Detect the corresponding proteins synthesized by the cells by appropriate methods. For s i R N A or antisense oligonucleotide samples, the knockout efficiency was analyzed by using NLP encapsulated with a control sequence as a control. In vivo gene transfection 14- Inject a small volume (~2,0 0 0 ul ) of N L P encapsulating 1 0 ~ 50ug of D N A or other oligonucleotides from the tail vein of the mouse. 15- At a suitable time (e.g. 24 h or 48 h ) , the animals are killed and the gene transfection efficiency in the target tissues is examined by appropriate methods. For example, to detect the transfection of fluorokinase gene in different organs: 丨. Execute the animal, remove the organ (e.g., liver, spleen, lungs, or heart), and wash it with cold phosphate buffer. 丨丨-Cut and transfer 200m g of tissue into a centrifuge tube containing Ig Zircon Beads (2. 4 mm diameter, Biospec products) and ImlIX Reporter Lysis Buffer (Promega). iii - Pound the tissue with a Mini-Beadbeater (Biospec product) at a beating rate of 5000 beating/m in for 30s. iV-Centrifuge the sample, take IOul of supernatant, and add 100ul of Fluorokinase Assay Solution to test for viability according to the kit instructions. For more product details, please visit Aladdin Scientific website.
D O PE can be dissolved in chloroform to prepare a 5 mg/ml stock solution.
Dilute to I X buffer before use.
IX TBS buffer contains 150 mmol/L NaCl, which is more suitable for internal administration than the low ionic strength IX T r is buffer.