Polyethylene glycol (peg) low molecular weight pei derivative, preparation method, use and complex thereof
A small molecular weight, polyethylene glycol succinimide carbonate technology, applied in the biological field, to achieve the effect of low surface charge density, low cytotoxicity, and small cytotoxicity
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Embodiment 1
[0056] Example 1. Preparation of Polyethylene Glycol (PEG) Polyethyleneimine (PEI) Derivatives (PEG-Et 1:1)
[0057] like figure 1 Shown, the preparation synthetic route schematic diagram of the PEGylated polyethyleneimine (PEI) derivative of the present invention, comprises the following steps:
[0058] (a) Dissolve PEI-Et and mPEG-SC with sodium bicarbonate respectively, and the two are made into 2mL and 5ml solutions respectively; PEI-Et and monomethoxypolyethylene glycol succinimide carbonate (mPEG- SC) was added into 0.1M sodium bicarbonate solution at a molar ratio of 1:1.
[0059] (b) At room temperature, the sodium bicarbonate solution of monomethoxypolyethylene glycol succinimide carbonate was added dropwise to the PEI-Et sodium bicarbonate reaction system, and the reaction was stirred for 4 hours;
[0060] (c) Separation and purification, the product is placed in a dialysis bag with a molecular weight cut-off of 3500Da and dialyzed for 48 hours (it can be any value...
Embodiment 2
[0071] Embodiment 2, the preparation of the complex (Polyplex) synthesized by PEG-Et 1:1 and plasmid
[0072] Weigh a quantitative polymer (PEG-Et 1:1), add ultrapure water to make a 2mg / mL solution, then filter it with a 0.45μm sterile filter head, and dilute the plasmid concentration to 20μg / mL;
[0073] Configure complex solutions with different mass ratios, keep the concentration of the plasmid solution constant, and then dilute the concentration of the polymer solution according to the mass ratio of PEG-Et 1:1 to the plasmid, and keep the concentration of the diluted polymer solution and plasmid solution. The volumes are equal, and finally the polymer solution is quickly added to the plasmid solution and mixed, and incubated at room temperature for 30-60 minutes, so that a series of complexes with a mass ratio can be obtained, which can be used for further determination of physical and chemical properties.
[0074] Polyplex particle size determination of PEG-Et 1:1 and pl...
Embodiment 3
[0083] The cell transfection experiment of the compound (Polyplex) synthesized by embodiment 3PEG-Et 1:1 and plasmid
[0084] In a 48-well cell culture plate, add 500 μL of cell suspension (MCF-7, CT-26 or HeLa) at a density of 5.0 to 10×10 4 / mL, cultivated overnight. When transfecting a 48-well plate, prepare the polymer into a 2mg / mL solution, filter it with a 0.45μm filter membrane, and dilute it to the required ratio according to the mass ratio of the sample to be tested and the plasmid, and then dissolve the polymer Add the substance solution to the plasmid solution, mix quickly, and incubate for 30 minutes. The volume of the complex added to each well is 50 μL, which is one-tenth of the total volume (500 μL), which meets the regulations. Three replicate holes were made for each quality ratio. For the positive control group, PEI 25kDa, 3 control wells were made based on the result of the best mass ratio of 2. During the incubation period, the cells were taken out of t...
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