Our research focuses on the development of cancer therapies using our iron oxide nanoparticle delivery platform. The advantage of our nanoplatform is its ability to cross the blood-brain barrier and accumulate in the tumor, delivering therapeutics. The goal is to add to the small arsenal of available therapies for glioblastoma.
The blood-brain barrier presents a challenge for the delivery of therapeutics to glioblastoma. Many compounds are unable to cross the barrier, making systemic administration of drugs for glioblastoma treatment near impossible. Currently available systemic treatments for this disease have many off-target toxicities.
Our nanoplatform overcomes this limitation by helping therapeutic moieties traverse the blood-brain barrier. The delivery of the therapeutic oligonucleotide to the tumor site can be monitored by various imaging modalities, making it a clinically relevant tool. The modular nature of the nanoplatform allows for tailor-made therapies to be developed, offering flexibility to address the vast molecular heterogeneity in glioblastoma subtypes.
This precision medicine approach with superior blood-brain barrier penetrance, allows us to expand upon limited therapeutic options. To begin, weigh the mouse and calculate the volume of magnetic nanoparticle, anti-microRNA-10b to administer for a dosage of 20 milligrams iron per kilogram in glioblastoma multiforme. Without introducing bubbles, prepare a 28-gauge syringe with the dose of magnetic nanoparticle anti-microRNA-10b.
Sterilize the mouse tail with a 70%isopropanol wipe. After drying, rotate the tail to position the lateral caudal veins at the top of the tail. Then insert the needle into the tail and pull back the plunger to establish a vacuum, continue inserting the needle until the blood enters the syringe.
Slowly inject magnetic nanoparticle solution and retract the needle after complete delivery. Apply pressure to the injection site with gauze until the bleeding stops. 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 autoexposure settings with a minimum signal threshold of 3, 000 counts, capture an image for the localization of luciferase-labeled U251 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 autoexposure 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*weighted images to visualize the nanoparticles.
With the following parameters, acquire a T2*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.
To begin, take the flash frozen brain stored in optimal cutting temperature compound out of 80 degrees Celsius storage and place it inside the cryostat chamber. Allow the samples to warm to the chamber temperature. Using a single-edged razor, cut the brain coronally at the injection site.
Mount the sample onto the specimen disc using a small amount of optimal cutting temperature. Apply ample pressure with the chilled weight inside the chamber for stable mounting of the sample. Move the mounted sample and specimen disc onto the specimen head.
Trim the tissue to the desired depth by taking 20-micrometer sections. Once the desired depth is reached, adjust the cryostat to take five to seven-micrometer sections of the sample. Then mount the sections onto the pre-labeled glass slide.
Fix the sections in the 4%paraformaldehyde for 15 minutes. After fixation, rinse the slide three times with DPBS. Add a 40-microliter drop of mounting medium containing DAPI onto the slide.
Carefully place a glass cover slip on top of the medium. Under fluorescence microscopy, visualize the tissue using DAPI and CY5.5 In ex vivo fluorescence microscopy of tumor tissues, the observed CY5.5 fluorescent signal confirmed the delivery of magnetic nanoparticles.
