The overall goal of this method is to synthesize a magnetic nanoparticle inside a protein cage and then to functionalize the protein such that it enables rapid attachment of the magnetic nanoparticle to cells. This method can be used to generate magnetic nanoparticles that enable rapid and efficient magnetic labeling of cells, which is important for applications such as MRI or magnetic cell separation. The main advantage of this technique is that cell magnetization can be achieved using low nanoparticle concentrations and short incubation times, which prevents potential adverse effects due to nanoparticle exposure.
To begin, deoxygenate 500 milliliters of deionized water by placing a tube connected to a nitrogen gas cylinder into the water and sealing the vessel with cling film. Then, bubble nitrogen gas through for approximately 60 minutes. Heat a water bath connected to the double-jacketed reaction vessel to 65 degrees Celsius.
Then, add 75 milliliters of 50 millimolar HEPES buffer, pH 8.6 into the reaction vessel. Seal the vessel and deoxygenate by bubbling nitrogen gas through the buffer solution for approximately 20 minutes. At the same time, stir the buffer solution using a magnetic stirrer.
After deoxygenating the HEPES buffer solution, remove the nitrogen tube from the buffer, keeping it suspended over the solution to maintain a nitrogen atmosphere. Add apoferritin to achieve a final concentration of 3 milligrams per milliliter. Continue the magnetic stirring, but reduce the stirring speed if foaming occurs.
For magnetoferritin synthesis, use two syringe pumps to simultaneously inject 10.1 milliliters of the iron-cobalt precursor, and 10.1 milliliters of the hydrogen peroxide into the apoferritin solution at a flow rate of 0.15 milliliters per minute. Continue with synthesis steps as described in the text protocol. Then, load the sample onto a column containing a cationic matrix using a peristaltic pump at a flow rate of 10 milliliters per minute.
Wash the column with approximately 100 milliliters of running buffer using a gradient pump at a flow rate of 10 milliliters per minute. To elute the protein, wash the column with 150 milliliters of increasing concentrations of sodium chloride and tris buffer at 10 milliliters per minute. As the protein elutes at a sodium chloride concentration of 500 millimolar, collect it in 50 milliliter fractions using an automated fraction collector.
Concentrate the 150 milliliters of magnetoferritin to a volume of approximately two milliliters using a 15 milliliter centrifugal filter unit followed by a four milliliter volume unit. Refer to the manufacturer's instructions of the centrifugal filter units for a detailed protocol of this procedure. Next, load the concentrated sample onto a gel filtration column using an injection loop.
Wash the column with running buffer at a flow rate of 1.3 milliliters per minute. Collect six milliliter fractions using an automated fraction collector. Protein monomers elute last.
At this point, purified magnetoferritin can be stored at four degrees Celsius until cationization. For 10 milligrams of magnetoferritin, weigh out 374 milligrams of DMPA and dissolve in 2.5 milliliters of 200 millimolar MES buffer. Adjust the solution pH to approximately seven using concentrated hydrochloric acid.
Toxic fumes are released when you adjust the pH of the DMPA solution with hydrochloric acid. Be sure to handle these materials in a fume hood. Add 2.5 milliliters of magnetoferritin solution at four milligrams per milliliter.
Add a magnetic stirrer and stir for two hours to equilibrate. After adjusting the solution to pH 5.0, add 141 milligrams of EDC powder to the DMPA magnetoferritin solution. Continue stirring for three and a half hours.
Filter the solution through a 0.22 micron syringe filter to remove any precipitates and dialyze the protein as described in the text protocol. Culture hMSCs as described in the text protocol. Wash the plated cells with two milliliters of room temperature PBS.
Then, add one milliliter of the sterilized cationized magnetoferritin solution to the plated cells before incubating for the desired time period. Wash the cells with PBS, and then harvest them by adding 0.5 milliliters of Trypsin-EDTA and incubating at 37 degrees Celsius for five minutes. After adding one milliliter of culture medium to inactivate the trypsin EDTA, transfer the solution to a 15 milliliter centrifuge tube and centrifuge for five minutes at 524 times G.Discard the supernatant and resuspend the cell pellet in 0.5 milliliters of magnetic separation buffer.
Next, attach the magnet to the multi-stand and add a magnetic separation column to the magnet. Place a pre-separation filter on the column. Then, add 0.5 milliliters of magnetic separation buffer to the pre-separation filter, and let it run through both the filter and the column to wash them.
Next, place a 15 milliliter centrifuge tube under the column and add 0.5 milliliters of the cell suspension to the filter reservoir of the magnetic separation column. When the reservoir is empty, add 0.5 milliliters of magnetic separation buffer. When the reservoir empties again, add another 0.5 milliliters of magnetic separation buffer.
Repeat the wash one more time, for a total volume of magnetic separation buffer of 1.5 milliliters. This wash step elutes all non-magnetized cells from the column. Remove the column from the magnet and place it in a fresh 15 milliliter centrifuge tube.
Then, remove the filter from the column reservoir. Add 1 milliliter of the magnetic separation buffer to the reservoir, and immediately push through the column using the plunger supplied by the manufacturer. This elutes the magnetized cells from the column into the centrifuge tube.
Proceed to perform iron quantification as described in the text protocol. Transmission electron microscopy images of negative-stained magnetoferritin samples showed that nanoparticles have formed inside the protein cage. Zeta potential measurements confirm that magnetoferritin obtained a positive surface charge after cationization.
Exposing human mesenchymal stem cells to cationized magnetoferritin for one minute resulted in the magnetization of 92%of the cell population and the delivery of 3.6 picograms of iron per cell. Increasing the incubation time to 15 minutes resulted in the magnetization of the entire cell population. After watching this video, you should have an understanding of how to synthesize a magnetic nanoparticle within the apoferritin cavity by sequentially adding metal salt precursors to the protein solution.
And then, how to chemically cationize the protein using TMPA coupling. Once mastered, magnetoferritin synthesis, purification, and cationization can be done in three days if it is performed properly. While attempting this procedure, it's important to avoid oxygen contamination and to maintain the correct temperature and pH throughout magnetoferritin synthesis.
Following on from this procedure, other cargoes like quantum dots, or therapeutic agents can be encapsulated in the cationized apoferritin cage to achieve more efficient delivery of those materials to cells. This technique can pave the way for researchers in the field of magnetic cell manipulation to explore magnetic labeling in cells that exhibit poor uptake of nanoparticles or that are very sensitive to prolonged nanoparticle exposure or elevated nanoparticle concentrations.
