The overall goal of this procedure is to synthesize bio functionalized Prussian blue nanoparticles for use of multimodal molecular imaging agents. This is accomplished by first synthesizing Prussian blue nanoparticles that contain either gadolinium or manganese ions, which enhance MRI signal. The second step is to attach fluorescent avadon onto the surface of the nanoparticles, which confers fluorescent imaging capability.
Next, the avadon coated nanoparticles are bio functionalized with the desired cell targeting biotinylated antibodies. A final step is to perform a quality control check on the bio functionalized nanoparticles by measuring their size zeta potential and temporal stability. Ultimately, the bio functionalized nanoparticles are utilized in multimodal molecular imaging applications to visualize a specific targeted population of cells within a mixture using fluorescence imaging and MRI.
Current commercial imaging agents are typically single mode and offer resolution only at the anatomical level. Prussian blue nanoparticles combines two complementary imaging modes MRI and fluorescence and allow for molecular imaging and sub-cellular levels. The applications of multimodal nanoparticles extend toward diagnosis of cancer and inflammatory diseases in addition to monitoring of their treatment responses.
This is because bio functionalized nanoparticles can target and bind into a population of cells within a specified region of the body. Although this method can provide insight into cancer and inflammatory disease diagnosis and response to treatment, it can also be applied to other pathological conditions that will benefit from the sensitivity and specificity of molecular imaging. Using fluorescence and MRI, We first had the idea for producing these nanoparticles when we determined that Prussian blue and FDA approved agent for human oral consumption could be stably synthesized using a one part synthesis scheme and robustly bio functionalized using standard bio conjugate techniques.
To begin, prepare a 5 million molar solution of potassium hexa sano ferrate two in five milliliters of deionized water to make solution A next repair solution B containing 2.5 millimolar iron three chloride and 2.5 millimolar manganese. Two chloride in 10 milliliters of deionized water for synthesis of manganese Prussian blue nanoparticles. Other nanoparticles can be synthesized as indicated in the text protocol transfer solution B to A round bottom flask and stir the solution at 1000 RPM at room temperature.
Using a peristaltic pump add solution, A dropwise into the vessel at a rate of 10 milliliters per hour. After the addition of solution A is complete, stir the mixture at 1000 RPM for an additional 30 minutes. Then transfer one milliliter aliquots of the mixture into micro centrifuge tubes and add 0.2 milliliters of five milli sodium chloride to each tube.
Centrifuge the tubes at 20, 000 times G for at least 10 minutes, and then carefully remove the supinate, leaving the pellet of nanoparticles. Next, add one milliliter of deionized water to each pellet. Use a micro tip to suspend the nanoparticles evenly in solution by sonication.
Wash the nanoparticles at least three more times to remove components of the initial reaction from the suspension after the final spin, re suspend the nanoparticles in one milliliter of deionized water after coating the nanoparticles with fluorescent den as described in the text protocol, remove the biotinylated antibody stocks from the refrigerator.Here. Use anti neuron glial antigen two and anti-human eotaxin three. Transfer the biotinylated antibody to a 0.2 micro nylon micro fuge filter, and then centrifuge the tube at 14, 000 times G for 10 minutes.
Next, add less than or equal to 0.05 milligram of the antibody per milligram of Aden coated nanoparticles and cover the tubes with aluminum foil to protect them from light. Incubate the tubes with gentle shaking for two to four hours at four degrees celsius. After attaching the antibodies, add 10 microliters of one milligram per milliliter nanoparticle sample to 990 microliters of deionized water in a disposable plastic vete cap the vete and V it to mix.
Well then determine the average size of the nanoparticles using a dynamic light scattering system with a measurement angle of 173 degrees. Start by preparing 10 milliliter suspensions of cells in PBS at a concentration of approximately 100, 000 cells per milliliter for mixed cultures tubes containing varying ratios of each cell line should be prepared as described in the text protocol at two milliliters of 5%BS, A to each tube to block the cells and to minimize non-specific binding. Then add one milliliter of one milligram per milliliter, bio functionalize nanoparticles to each tube, and incubate the mixture for one hour.
Next, rinse the cells free of unbound nanoparticles by spinning the tubes at 1000 times G for five minutes and re bending the pellets in one milliliter of PBS. Repeat this process at least twice more. Fix the cells using 10%formaldehyde in PBS and then add 10 micrograms per milliliter, seven amino actin and mycin D in PBS to stain the cells, incubate the tube on ice for 30 minutes and then rinse the cells with PBS.
Next, load the sample into the flow cytometer and analyze 10, 000 gated cells from each sample. A similar procedure to image cells bound to fluorescent nanoparticles using laser confocal microscopy is described in the text protocol. To begin, wear the cells in T 75 flasks to approximately 80%confluence, and then rinse the cells free of medium with five milliliters of PBS.
Add five milliliters of 1%BSA in PBS and block the cells for one hour. Then add five milliliters of 0.5 milligrams per milliliter antibody coated nanoparticles to the flask and incubate the cells for one hour to allow the nanoparticles to bind. Next, rinse the cells three times with five milliliters of PBS to remove unbound nanoparticles.
Then add two milliliters of 0.25%tripsin EDTA solution, and incubate the cells for five minutes to detach the cells from the flask. Add eight milliliters of DMEM to quench the tryin and then transfer the cell suspension to centrifuge tubes. Spin them at 1000 times G for five minutes to pellet the cells.
Next, aspirate the supinate and resuspend the cells in one milliliter of PBS. Add one milliliter of 10%formaldehyde in PBS to fix the cells and then transfer 100 microliters of each sample into separate wells of a 96 well flat bottom plate. Add 100 microliters of molten 1%aros in deionized water to each well and mixed by pipetting up and down.
Allow the gel to solidify at four degrees Celsius for 12 hours to yield a phantom. After the gel solidifies, place the phantom in a horizontal three T clinical magnet next to a solid 150 centimeter cube block of 2%agar. Then secure the phantom and the agar block within the center of an eight channel HD brain coil to measure the relaxation times use 0.5 millimeter thick coronal slices taken at mid height of the 96 well plate dynamic light scattering assays were used to determine the hydrodynamic diameter of the generated nanoparticles stability Studies verified the long-term stability of the nanoparticles are used in both water-based and cell media-based experiments.
Laser confocal microscopy showed fluorescent nanoparticles loaded with antibodies for EOT three found specifically to the surface of EOL one cells. When antibodies for a NG two are used, they specifically bound to the surface of BSG neurospheres. The bio functionalized nanoparticles were able to successfully fluorescently label a population of targeted cells as measured by flow cytometry.
Additionally, the nanoparticles were able to target a specific subpopulation of cells in a mixture even at low target cell to control cell ratios. Finally, bio functionalized nanoparticles increased the MRI contrast of targeted cells, cells treated with nanoparticles loaded with a nng. Two antibodies showed hyperintensity in the T one weighted sequences compared to cells treated with control particles.
In contrast, the A NNG two nanoparticles conferred hypo intensity in the T two weighted sequences compared to the control particles. Once masters. The synthesis of bio functionalized presion blue nanoparticles can be completed within two days if it is performed properly.
Similar to this procedure, the nanoparticles can be bio functionalized with different biopolymers and targeting ligand to investigate new disease and new conditions. After watching this video, you should have a good understanding of how to synthesize Prussian blue nanoparticles for fluorescence and imaging applications to label population of cells. Don't forget to rigorously follow the safety guidelines and standard operating procedures for conducting the studies in the clinical MRI magnet.
