This video introduces a fast and efficient method for the synthesis of magnetic nanoparticles and the use of magnetometry to photograph transformation in the biological environment. Magnetic nanoparticles have attracted increased attention for biomedical applications, And it is important to understand that fate once internalized in cells. This is achieved here by measuring the cellular magnetism.
For both magnetic nanoparticles synthesis and measurements, result could be affected by contamination. Use well-cleaned chemical vials and make sure that the magnetometer equipment is free of contamination. The magnetometry samples holder is usually used for powder samples.
We show how to adapt the use of the holder to liquid or biological samples. In a 30 milliliter mana wave glass vial dissolve 400 milligrams of iron three acetylacetonate in 10 milliliters of benzyl alcohol. Using a microwave reactor, heat the suspension from 25 degrees Celsius to 250 degrees Celsius at a rate of 11.25 degrees Celsius per minute, and maintain it at 250 degrees Celsius.
To separate the nanoparticles, apply a neodymium magnet for 30 minutes. To pellet the nanoparticles, perform a series of washes using 10 milliliters of the designated wash fluid and applying a neodymium magnet for five minutes. Remove the filtrate and add 12 milliliters of 10 to the minus two molar hydrochloric acid.
Centrifuge the suspension and a 100 kilodalton filter at 2, 200 times G for 10 minutes. Then resuspend the nanoparticles in 10 milliliters of 10 to the minus two molar hydrochloric acid. Prepare the coating molecule solution as described in the manuscript.
Add the 10 milliliter nanoparticle aqueous dispersion to the coating molecule solution at a mass ratio of five to one between the coating molecules and the nanoparticles. Then agitate the mixture for two hours at room temperature using a magnetic stirrer. After the reaction is complete, adjust the pH of the mixture to seven using one molar sodium hydroxide.
When the culture of human mesenchymal stem cells are at passage five and 90%confluent, remove the medium and rinse the cells with serum-free RPMI without glutamine. To each 150 square centimeter culture flask, add 10 milliliters of the nanoparticle suspension. Incubate the flasks at 37 degrees Celsius and 5%carbon dioxide for 30 minutes.
Then discard the medium containing the nanoparticle suspension and wash the cells once with serum-free RPMI 1640. Add DMEM supplemented with 10%FBS and 1%penicillin streptomycin. Incubate overnight at 37 degrees Celsius and 5%carbon dioxide to allow complete internalization of the nanoparticles.
Add 10 milliliters of 0.05%trypsin EDTA prewarmed for 37 degrees Celsius to each flask of magnetically labeled MSCs. After two to three minutes, when the cells are detached, immediately inactivate the trypsin by adding one third of the volume of pre-warned supplemented DMEM. Centrifuge the detached cells at 260 times G for five minutes.
Aspirate the medium and resuspend the cells in a small volume of cell-spheroid culture medium at a concentration of approximately 200, 000 cells per 50 microliters. Add one milliliter of freshly prepared cell-spheroid culture medium to a 15 milliliter sterile conical centrifuge tube. Then add a volume of cell suspension corresponding to 200, 000 cells.
Centrifuge these magnetically labeled cells at 180 times G for three minutes in order to form a cell pellet at the bottom of the tube. Then place the tube in the incubator at 37 degrees Celsius and 5%carbon dioxide, keeping the cap slightly opened for gas exchange. Place either a given volume of magnetic nanoparticle or a fixed single cell-spheroid suspension into the vibrating sample magnetometer, or VSM, sample holder.
If necessary, PTFE tape can be used to prevent any leakage. Insert the sample holder into the VSM and scan for sample offset. Place the sample at the position corresponding to the magnetization maximum.
Perform the first measurement at a low magnetic field between negative 1, 500 Oersteds and positive 1, 500 Oersteds at a rate of 20 Oersteds per second. Perform a second measurement at a high magnetic field between zero Oersteds and positive 30, 000 Oersteds at a rate of 200 Oersteds per second. 10 microliters of nanoparticles dispersed in an aqueous solution were measured in the vibrating sample magnetometer.
The slope of the second part of the curve is the diamagnetic constant, representing the diamagnetic signal from the water and the sample holder. This diamagnetic constant was subtracted from the magnetic moment of the solution to obtain the magnetic moment of the nanoparticles only. The magnetic moment at saturation was then determined.
Coated nanoparticles were internalized in stem cells. Electron microscope images show the citrate-coated and PAA-coated nanoparticles confined in the endosomes. After the cells were pelleted by centrifugation, they formed a cell-spheroid that can be kept in culture for extended time periods.
Individual cell-spheroids were also measured using the VSM and the saturation magnetic moment was used to calculate the quantity of nanoparticles in a cellular sample. Uptake of the nanoparticles was found to depend on their incubation concentration. A decrease in magnetism of the cell-spheroids over time indicated the biodegradation of the nanoparticles, which was confirmed by electron microscopy.
Degradation was more substantial in the citrate-coated nanoparticles than in the PAA-coated nanoparticles. Using magnetometry, the degradation of magnetic nanoparticles within cells can be precisely quantified, and the impact of the futures of nanoparticles on the interested in raw behavior can be assessed. In this protocol, the magnetometry measurements were performed on cell-spheroids, but they can be extended to cells into suspension or to organs.
With the described method, you are able to explore the interactions of newly designed magnetic nanoparticles in different cell system of interest, but also you can follow their in vivo biodistribution and fate.
