24 hours after the magnetic nanoparticle, anti-microRNA 10b injection, weigh the mouse and calculate the volume of d-luciferin to administer for a dosage of 150 milligrams per kilogram. Prepare a 28 gauge syringe with the volume protecting the solution from direct light. Once anesthetized, gently scruff the mouse to avoid further injuring the surgical site, and inject the dose of d-luciferin intraperitoneally.
Allow the mouse to recover and wait for 10 minutes before imaging. After preparing an in vivo imaging system, or IVIS scanner, place down the black low-fluorescence mat on the imaging stage and configure the nose cone array for imaging. In the software, click imaging wizard.
Then, select bioluminescence imaging, followed by open filter. On the next screen, select imaging subject and field of view. Place the anesthetized mouse in a prone position on the IVIS scanner.
Click acquire sequence, and using auto exposure settings with a minimum signal threshold of 3000 counts, capture an image for the localization of luciferase-labeled U-251 glioblastoma cells. Next, in the imaging wizard, select fluorescence, followed by filtered pair with epi illumination. In the next screen, select the probe, Cy5.5.
Select the imaging subject and field of view. Using auto exposure settings with a minimum signal threshold of 6, 000 counts, capture an image for the localization of magnetic nanoparticles, anti-microRNA 10b. Place the anesthetized mouse prone on the MRI bed.
Immobilize and position the head for scanning using a bite bar and ear bars. Install the lubricated rectal temperature probe and ensure respiration and temperature monitoring are functioning. Position the mouse brain coil over the head by fitting the pegs on the mouse bed into the holes on the coil, and tape the coil into place to reduce movement during scanning.
Place a small warm water circulating pad over the top of the mouse to maintain body temperature. Move the mouse and imaging bed into position for scanning. In the acquisition software, start the wobble setup step to tune and match the MRI coils.
Ensure that the trace is centered and as deep as possible. Acquire a three plane localizer scan of the brain. Using the following parameters, acquire two-dimensional T2 weighted scans to detect the tumor.
Acquire a b0 map of the whole brain to calculate a localized shim using the map shim utility. Use three dimensional T2 star-weighted images to visualize the nanoparticles. With the following parameters, acquire a T2 star map for further nanoparticle imaging using the two dimensional T2-weighted image as a reference to position the scan over the tumor.
Acquire 10 positive echo images with five milliseconds of echo time spacing. At the experimental endpoint, weigh the mouse, and calculate the volume of d-luciferin to administer for a dosage of 150 milligrams per kilogram. Conduct live bioluminescence imaging 10 minutes post-injection, as previously described.
Place the Petri dish with the excised brain from the euthanized mouse in the IVIS scanner. Image the brain with both bioluminescence and fluorescence modalities using the same acquisition settings as the in vivo imaging. The Cy5.5 fluorescence and bioluminescence signals in magnetic nanoparticles anti-microRNA 10b-injected mice showed clear colocalization, indicating delivery of the nanoparticles to the tumor, while control mice showed no fluorescence signal.
Ex vivo fluorescence imaging showed the localization of the nanoparticles in major clearance organs, such as the liver and kidneys. A characteristic decrease in T2-weighted MRI signal demonstrates the potential of magnetic nanoparticles, anti-microRNA 10b as an MRI contrast agent to cross the blood-brain barrier and its accumulation in the tumor region.
