This measure can help answer key questions. In the field of humanology. That answer in the function of macrophages under physiological end to pathological conditions.
The main advantage of this technique is that polyethyleneimine coated can efficiently en vitro without compromising cell. Under optimal doses. The implications of this technique extend toward therapy and diagnosis of many chronic inflammatory disorders and cancers.
Where macrophages play essential role in the pathogenesis. Demonstrating the procedure will be Na Jia. A graduate student from my lab.
And Kaixuan Wang a graduate student from Dr.Xiaohua's lab. To prepare allylic acid modified super paramagnetic iron oxide nanoparticles or SPION's first add 28 grams of pharas chloride hexahydrate. And 20 grams pharas selfate heptahydrate to a beaker containing 80 milliliters of deionized water.
And use a glass conduit to introduce nitrogen into the solution. Stirring until the solid matter has dissolved. Heat the reaction mixture to 72 degrees Celsius with stirring at 800 rotations per minute.
And add 40 milliliters of 28%ammonia water to the beaker. After five minutes add nine milliliters of allylic acid drop wise. And maintain the mixture at 72 degrees Celsius for three hours with continuous stirring.
At the end of the incubation cool the resulting solution to room temperature. And precipitate the mixture via magnetic separation according to standard protocols. Then wash the SPION containing precipitate three times with absolute ethyl alcohol.
And asperse the precipitate in 100 milliliters of n-hexane. To prepare dimercapto acrylic acid modified SPION's add 800 milligrams of the allylic acid modified SPION's. Dispersed in 200 milliliters of n-hexane and 400 milligrams of dimercapto acrylic acid dispersed in 200 milliliters of acetone into a three neck flask in a water bath at 60 degrees Celsius.
Next add 200 microliters of triethylamine drop wise to the flask with stirring at 1000 rotations per minute and refluxing. A black precipitate can be obtained by magnetic separation after five hours. Then use tetramethylammonium hydroxide to adjust the pH of the solution for homogenous dispersal's of the hydrophilic SPION's in deionized water.
Degenerate polyethyleneimine coated SPION's add the dimercapto acrylic acid modified SPION's colloidal solution drop wise into a 10 kilodalton polyethyleneimine solution. In a 500 milliliter three neck flask under mechanical stirring at 1000 rotations per minute for two hours. At the end of the incubation add the resulting solution to an ultra filtration tube with a molecular weight cut off of 100 kilodaltons and a content of 15 milliliters.
Centrafuse the sample until the remaining solution reaches a one milliliter volume. Add deionized water to the solution to bring the volume back to 15 milliliters. And centrafuse and rehydrate the solution ten more times as just demonstrated.
Then filter the solution through a 22 micron filter for storage at four degrees Celsius. To prepare the siRNA nanoparticles first add three microliters of freshly prepared 20 micromolar siRNA solutions to five labeled RNA's free microcentroafused tubes. And add 0, 8, 1.6, 3.2, and 6.4 micrograms of polyethyleneimine SPION's to tubes labeled zero, one, two, four, and eight respectively.
After mixing with gentle pipetting, incubate the samples for 30 minutes at room temperature to allow the polyethyleneimine SPION siRNA complexes to form. And prepare a three percent agarose gel with I purity agarose. At the end of the incubation add one microliter of 6X DNA loading buffer per five microliters of sample to each tube and mix carefully.
Then load all of the samples onto the gel. And run electrophoresis at five volts per centimeter. Until the bromophenol blue dye migrates two thirds of the length on the gel.
One day prior to the transfection wash a mouse microphage like RAW264.7 cell culture with PBS. And treat the cells with one milliliter of 25%trypsin for five to ten minutes at 37 degrees Celsius in a five percent carbon dioxide incubator. When the majority of the cells have detached.
Inactivate the trypsin with five milliliters of complete DMEN. Then pippete the solution several times to disperse any cell clusters. Transfer the cell suspension to a sterile 15 milliliter conackel tube for centrifugation.
Then re suspend the cells in five milliliters of fresh complete DMEN for counting. Dilute the cells to a 4.5 times ten to the fourth cells per milliliter of complete DMEN concentration. And add two milliliters of medium to each well of a six well plate for a 24 hour incubation in the cell culture incubator.
The next day prepare the appropriate ratio of polyethyleneimine SPION siRNA nanoparticles in a 1.5 milliliter RNA stream microcentrifuge tube with gentle mixing as just demonstrated. And add the appropriate volume of polyethyleneimine SPION siRNA nanoparticle complex to each well which has been replaced with one milliliter of fresh medium. Prepare polyethyleneimine SPION si complexes.
in this instance many more months of polyethyleneimine SPION can be used Thus minimizing their potential toxicity. Then swirl the plate carefully to achieve an even distribution of the nanoparticles across the well bottoms. Return the plate to the cell culture incubator until subsequent cellular update or gene knockdown deficiency analysis.
Polyethyleneimine SPION's with a zeta potential of 30.5 and 37 millivolts do not exhibit an apparent cyto toxicity at concentrations up to 30 micrograms of iron per milliliter. Which is about two fold hire than the concentration normally used for cell transfection. Polyethyleneimine SPION's zeta potential of 48 millivolts however are toxic.
Even at the lowest dose examined. As analyzed by flow cytometry, more than 90%of cells can be transfected with fluorescently labeled polyethyleneimine SPION siRNA complexes at 15 micrograms of iron per milliliter. Assessment of the effects of polyethyleneimine SPION siRNA concentrations on cellular internalization by crushing blue staining reveals minimally detectable staining at 7.5 micrograms of iron per milliliter.
But clearly visible spots at 15 micrograms of iron per milliliter that do not increase at higher concentrations of iron likely due to polyethyleneimine SPION siRNA uptake saturation. Peritoneal macrophages transfected with polyethyleneimine SPION's harboring specific siRNA exhibit a significant decrease in targe mRNA levels compared to nonspecific siRNA. Suggesting that siRNA can escape from endocytic vesicles into the cytoplasm to reach the RNA interference machinery.
Further after intravenous injection of a single dose of polyethyleneimine SPION siRNA nanoparticle complexes flocentimetrical analysis of CD11b positive and CD3 positive cells reveals a more efficient uptake of the nanoparticles by CD11b expressing macrophages than by CD3 positive cells at any time point in all of the organs examined. While attempting this procedure it's important to remember to prepare the polyethyleneimine SPION's with an average data potential not higher than 37 millivolts and to prepare the polyethyleneimine SPION siRNA complexes on the low iron to siRNA ratios. This technique compares the need for researchers to explore therapeutic potential of targeting macrophage in animal motors of human disease such as and cancer.
