Chemistry
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Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery
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Summary August 6th, 2019
This work presents the preparation of methionine functionalized biocompatible block copolymers (mBG) via the reversible addition-fragmentation chain transfer (RAFT) method. The plasmid DNA complexing ability of the obtained mBG and their transfection efficiency were also investigated. The RAFT method is very beneficial for polymerizing monomers containing special functional groups.
Transcript
Cationic polymer drug carriers have the advantages of good stability, low immunogenicity, and facile preparation and modification. Here, we offer a rapid and simple method for its synthesis. Reversible addition-fragmentation chain transfer polymerization method is a method applicable to yield block polymers with controlled molecular weight and structure and carrying functional groups.
These drug carriers can simultaneously carry different drugs to achieve the collaborate treatment of cancer, inflammation, and other diseases. IFT polymerization is a classic method, and this protocol gives an example of its application in gene therapy. The key to the success for synthesis of this polymer is to precisely control the reaction temperature.
It is more advisable to use an oil bath than a water bath. To begin this procedure, obtain a round-bottom flask with a rubber stopper and a magnetic stirrer to be used as the polymerization bottle. Dissolve 1.87 grams of bis-hydroxy HEMA in one milliliter of distilled water in the polymerization bottle.
In a five-milliliter beaker, dissolve 0.03 grams of CTP and 0.02 grams of ACVA in 0.5 milliliters of 1, 4-dioxane. Add this mixture to the polymerization bottle. Then, use a condensate trap to freeze the solution in the polymerization bottle.
Fix the bottle to the iron support, and use a conduit tripped with a needle to vacuumize and inject nitrogen into the reaction mixture. Then, seal the polymerization bottle, and thaw the solution at room temperature for 30 minutes. Repeat this freeze-pump-thaw cycle three times.
After this, place the bottle into an oil bath at 70 degrees Celsius and let the solution react for 24 hours under the nitrogen atmosphere. The next day, chill the polymerization bottle at zero degrees Celsius and open the rubber stopper to terminate the polymerization reaction. Next, precool acetone in a refrigerator at negative 20 degrees Celsius for two hours, and mix it with the reaction solution in the polymerization bottle at a ratio of 50 to one.
Centrifuge this mixture at 8, 200 times g for 10 minutes to remove the acetone and collect the precipitate. To purify the synthesized poly bis-hydroxy HEMA, dissolve the collected precipitate in two milliliters of pure water. Then, mix it with 100 milliliters of precooled acetone at a ratio of one to 50.
Centrifuge the solution at 8, 200 times g for 10 minutes to collect the precipitate. Repeat this purification process of dissolving, mixing, and centrifuging the precipitate three times. First, dissolve 0.96 grams of APMA and 0.93 grams of the purified poly bis-hydroxy HEMA in five milliliters of distilled water in a 10-milliliter beaker.
Dissolve 0.01 grams of ACVA in 0.5 milliliters of 1, 4-dioxane, and add this to the APMA and poly bis-hydroxy HEMA solution. Transfer this mixture to a polymerization bottle, and ventilate with dry nitrogen for one hour. Then, put the polymerization bottle into an oil bath at 70 degrees Celsius, and let it react for 24 hours under the nitrogen atmosphere.
The next day, chill the polymerization bottle at zero degrees Celsius and open the rubber stopper to terminate the polymerization process solution. To begin, seed MCF-7 cells into the wells of a six-well plate at a density of 20, 000 cells per well. Culture the cells in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for 12 hours.
After this, replace the culture medium with the fresh culture medium containing cationic polymer and pDNA polyplex polyplexes of different ratio of nitrogen-to-phosphorus ratios. Culture the cells for six hours, and then replace the medium with two milliliters of fresh RPMI 1640 medium. Continue culturing for 48 hours.
Then, collect the cells, and use a flow cytometer to detect the green fluorescence. In this study, methionine-functionalized biocompatible block copolymers are prepared via the reversible addition-fragmentation chain transfer method, and the plasmid DNA complexing ability of the obtained mBG and their transfection efficiency is investigated. The average particle size and zeta potential of mBG/pDNA polyplexes are 124 nanometers and plus 15.7 millivolts, respectively, at an N-to-P ratio of 16.
Electrophoretic retardation reveals that pDNA with an N-to-P ratio of zero can move without restraint in the agarose gel and is observed as a stripe in the gel imager. When pDNA is complexed with mBG, the movement of pDNA is retarded, and subsequently, the brightness of the band is reduced. This shows that mBG can completely complex the pDNA when the N to P is higher than four.
The cytotoxicity of the mBG/pDNA polyplexes is then measured using the standard MTT assay These results show that mBG/pDNA polyplexes have cytotoxicity than PEI/pDNA polyplexes at the N-to-P ratios of four, eight, 16, and 32. As can also be seen, the cytotoxicity of the mBG/pDNA polyplexes increases with increased N-to-P ratio. This increased cytotoxicity is a result of the positively charged GPMA component.
The N-to-P ratio used in the pDNA transfection experiment is selected according to the cytotoxicity results. The transfection abilities of mBG1, mBG2, and mBG3 are compared by measuring the GFP fluorescence intensity, and the results showed that mBG3 is the optimal gene carrier. It is important to exhaust air from the silt round-bottom flask and maintain the reaction temperature.
The obtained polymer drug carriers can be applied to the co-delivery of chemotherapy drugs and gene drugs to synergistically treat drug-resistant cancer. The molecular mechanism of combination of gene drugs and chemotherapeutic drugs can be further explored for the treatment of drug-resistant tumors. Acetone is rated as low toxic for its acute toxicity.
Keys, inhalation or contact with eyes or skin should be avoided. Avoid to the risk of scalding during the heating procedure.
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