PEGylated nanoparticles of poly(L-lysine) and DNA
PEGylated nanoparticles of poly(L-lysine) and DNA
Nanoparticles of P E G-coated poly(L-lysine) and D N A are water-soluble, stable in saline and tissue fluids, transfect undivided cells (Liu et al. 2003), show little toxicity (Ziady etal. 2003b), and are effective in animals as well as in humans (K o nstanetaL 2004). They are simple, reliable and reproducible in their preparation. These properties also represent the fundamental advantages of nonviral gene vectors. Author: T. Friedman et al., Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Preparation and analysis of PEGylated poly(L-lysine)/D N A nanoparticles Move Preparation and analysis of PEGylated poly(L-lysine)/D N A nanoparticles Material: PEGylated poly(L-lysine) / D N A nanoparticles reagents C K 30 This peptide, which is cysteine at the amino-terminus and contains 30 lysine residues, is usually synthesized by solid-phase trifluoroacetate synthesis and characterized by high-performance liquid chromatography (HPLC), mass spectrometry, and quantitative amino acid analysis.1 The peptide has a high purity, usually greater than 95 % (CK28-CK31 content), and a dimer content of less than 1 %. The purity is usually greater than 95% (CK28-CK31 content) and the dimer content is less than 1%. Rapid protein liquid chromatography and quantitative 4,4-dithiodipyridine analysis of the lyophilized peptides after one year of storage at 20°C under argon protection showed that the purity remained above 90%. Dextrose (5 %) or NaCl (0.9 %) Dimethyl sulfoxide (DMSO) P E G monomethyl ether maleimide derivative (m P E G -M A L -10k ) (molecular mass I OkDa) mPEG-MAL-I O k is available from Nektar Therapeutics. The molecular mass and dispersion were determined by gel permeation chromatography. The 80 % PEG chain of the above material should have a functionalized maleimide group attached to the end of the chain, which can be verified by 1 H-NMR analysis. The impurity content (PEG dimers) should be less than 10 %. Experimentally, 5 kDa or 20 kDa PEG can also be used. Phosphate buffer (PBS) (0.lmol/L, p H 7.2) / EDTA (5m m o l /L) (recipe from Sama-brook and Russell 2001). Plasmid D N A (0.2m g /m l aqueous solution) The plasmid was amplified by bacteria and purified by alkaline lysis followed by CsCl-ethidiumbromide gradient centrifugation. The plasmid was then precipitated twice with 0.1 v/v of 3 ml/L sodium acetate and 2.5 v/v of ethanol and resuspended in water, and the concentration was determined by UV spectrophotometry. The obtained D N A was analyzed by R N a s e A < ! > and R N a s e T l twice and then resuspended to a final concentration of 1.5 to 2 mg/ml. It should be ensured that there is no bacterial chromosomal DNA or RNA contamination during plasmid preparation, and that the open-loop and linear DNA content should be less than 30%. Sterilized water for injection This can be purchased from Baxter. It is used to prepare aqueous solutions of DNA and PEGylated peptides. Trifluoroacetic acid (0.1 %) or 50 mM o l/L ammonium acetate. Instrument. Agarose gel and electrophoresis apparatus Dialysis equipment Dextran G l 5 column Spectrophotometer transmission electron microscope Methods 1. C K 3 was tested by the 4 ,4'-dithiobisheptane method (Grassetti and Murray 1967). The content of reactive hydrophobic groups in the The expected value of the content of active sulfhydryl groups is calculated on the basis of the mass of the peptide, its purity, and the water ' content, which should generally be not less than 8 0 % . 2- Dissolve 60 um ol CK 3. (trifluoroacetate) in 15m l 0.1m o l/L PB S ( p H 7.2) /5p m o l/LE D T A . 3 . Dissolve 66 umol of mPEOMAL-10k in 15 ml of DMSO. vortex at room temperature and add dropwise to the above CK 3 solution within 5 m in, and continue stirring for an hour. For this reaction mPEG-MAL-lOk should be overdosed by 10% (based on the activity of the maleimide). At a p H of 7, maleimide reacts 1000 times faster with sulfhydryl groups than with amino groups (Hermanson 1996). 4 The sulfhydryl content was tested by the 4, 4'-dithiodipyridine method to ensure complete reaction. C K 3. The P E G grafting rate should be close to 1 0 0 % . The PEGylated CK3 product described above should only be used if more than 90% of the sulfhydryl groups have been reacted. 5. Purify the reaction product with dextran gel G 15, equilibrating the gel column with 0.1 % trifluoroacetic acid or 50 mM l/L ammonium acetate. The gel column was equilibrated with 0.1 % trifluoroacetic acid or 50 mM l/L ammonium acetate. The peptide-containing effluent fraction was identified at 220 nm, collected and lyophilized. The lyophilized PEGylated polylysine can be stored at 1 20°C for at least 2 years. Preparation of nanoparticles 6 . Resuspend CK38P E G lO k with water to give a final concentration of 7.1m g /m l (trifluoroacetate) or 6.4m g /m l (acetate). 7. aliquot 0 -9m l D N A (0-2m g / m l of aqueous solution), IOOfil each, to a resuspension of ○ - Iml C K 30P E G l O k , and shake for 2m i n at room temperature. The final DNA concentration was 0.18 mg/ml, and the positive to negative charge ratio was 2 : 1 (NH3+/PO4- ). The above samples were dialyzed at 8.4°C against 5% dextrose or 0.9% NaCl solution to remove free C K 3eP E G l O k and unreacted P E G . Analysis of nanofrequency particles 9- The morphology of the particles was observed by transmission electron microscopy. The nanoparticles produced need to fulfill the following specifications: the nanoparticles are not aggregated , electron dense, elliptical in shape when trifluoroacetic acid is the counterion and rod shaped when acetic acid is the counterion; the particle size of the particles should be the same as the plasmid particle size. 10. Agarose gel analysis of particles. No free or degraded D N A should be detected, and the particles should remain in the sample well or move forward only slightly. Pellets treated with 75% mouse serum for 2 h at 37°C, followed by 2.5% trypsin for 40 m in at room temperature, or incubated with DNA, should have greater than 95% of DNA structurally intact, although the superhelical DNA may be severed. 11. Stability of particles in saline. At room temperature, centrifuge the pellet at 3400 g. The ratio of the absorbance value of the supernatant at 260 nm (A 2 w ) to the starting pellet suspension A 26. should be (1 ± 10)%. 12. D N A turbidity determination (Ziady etal. 2003a). The apparent absorption value (vertical coordinate) of the particulate suspension was plotted against the wavelength . The apparent absorption values of the particulate suspensions (vertical coordinates) were plotted against the wavelength (330-415 n m, horizontal coordinates) in logarithmic coordinates, and the slopes of the straight lines should be in the range of 3.5 to 4.5. For more product details, please visit Aladdin Scientific website.
Preparation of P E G-chemical polylysine (C K 3QP E G 1 0 k )