PEGylation low molecular mass PEI derivative, preparation method and application thereof, and its compound
A low molecular weight, polyethylene glycol succinimide carbonate technology, applied in the biological field, to achieve low cytotoxicity, simple structure, and low surface charge density
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[0056] Example 1. Preparation of polyethylene glycol (PEG) polyethyleneimine (PEI) derivative (PEG-Et 1:1)
[0057] Such as figure 1 As shown, the schematic diagram of the preparation synthetic route of the PEGylated polyethyleneimine (PEI) derivative of the present invention includes the following steps:
[0058] (a) Dissolve PEI-Et and mPEG-SC with sodium bicarbonate respectively, and make 2mL and 5ml solutions respectively; combine PEI-Et and monomethoxy polyethylene glycol succinimide carbonate (mPEG- SC) The molar ratio is 1:1 and added to 0.1M sodium bicarbonate solution.
[0059] (b) At room temperature, add the sodium bicarbonate solution of monomethoxy polyethylene glycol succinimide carbonate dropwise to the PEI-Et sodium bicarbonate reaction system, and stir for 4 hours;
[0060] (c) Separation and purification. The product is placed in a dialysis bag with a molecular weight cut-off of 3500Da for 48 hours (any value from 24 to 48 hours). After the dialysis is completed, the...
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[0071] Example 2. Preparation of PEG-Et 1:1 and plasmid synthesis complex (Polyplex)
[0072] Weigh a quantitative polymer (PEG-Et 1:1) polymer, add ultrapure water to prepare a 2mg / mL solution, then filter with a 0.45μm sterile filter, and dilute the plasmid concentration to 20μg / mL;
[0073] Configure the complex solution with different mass ratios to keep the concentration of the plasmid solution unchanged, and then dilute the concentration of the polymer solution according to the mass ratio of PEG-Et 1:1 to the plasmid to keep the diluted polymer solution and plasmid solution The volume is equal, and finally the polymer solution is quickly added to the plasmid solution and mixed, and incubated at room temperature for 30 to 60 minutes, so that a series of mass ratio complexes can be obtained, which can be used for further physical and chemical properties determination.
[0074] PEG-Et 1:1 and plasmid synthesis complex (Polyplex) particle size measurement
[0075] The sample size fo...
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[0083] Example 3 Cell transfection experiment of PEG-Et 1:1 and plasmid synthesis complex (Polyplex)
[0084] In a 48-well cell culture plate, add 500μL of cell suspension (MCF-7, CT-26 or HeLa) with a density of 5.0~10×10 4 / mL, incubate overnight. When transfecting a 48-well plate, configure the polymer to a 2mg / mL solution, and filter it with a 0.45μm filter membrane aseptically, dilute it to the required ratio according to the set mass ratio of the sample to be tested to the plasmid, and then polymerize Add the substance solution to the plasmid solution, mix quickly, and incubate for 30 min. In this way, 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. Each mass ratio makes 3 replicate holes. Positive control group PEI 25kDa, with its best mass ratio of 2, make 3 control wells each. During this period of incubation, remove the cells from the incubator, remove the medium with serum, and use 2...
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