Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We're developing minimally invasive contrast agent, microbubble based therapeutic approaches in which the permeation and or ablation of the micro vasculature may be controlled by varying ultrasound pulsing parameters. Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through chemotherapeutic drug delivery and microvascular ablation. Here, ultrasound activated microbubbles are used to target the delivery of nanoparticles, anti mechanically ablate tumor tissue for drug delivery.

Nanoparticle drug carriers are fabricated and drug carriers may be co injected with microbubbles or conjugated to the shells of microbubbles. As shown here, ultrasound is then targeted to the tumor, and as microbubbles pass through the targeted region, they expand and then collapse perme the surrounding micro vasculature and enhancing payload delivery. The effectiveness of drug targeted treatment can be assessed by quantifying the biodistribution of nanoparticles.

Furthermore, in many brain cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of cavitation induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis without significant heating. These results may have bearing on the development of transcranial high-intensity focused ultrasound treatments for brain tumors that are not amenable to thermal ablation.

The main advantage of our technique over existing methods such as thermal ablation, surgical resection, and radiation therapy, is that our technique is targeted non-invasive and has the potential to enhance payload delivery. This treatment approach may alleviate many of the technical difficulties associated with transcranial high intensity focused ultrasound. These difficulties include off target thermal accumulation, incomplete tissue ablation, and inability to treat tumors close to the skull, as well as extended treatment duration.

To evaluate the effectiveness of targeted drug delivery and ablative treatments, mice are injected with glioma tumor cells. After tumors have grown for 12 days, ultrasound treatment has performed to either target delivery of nanoparticles or mechanically ablate tumor tissue. Begin this protocol by preparing the nanoparticles that will be used for targeted drug delivery studies.

Prepare a 2%polyvinyl alcohol or PVA solution by dissolving 20 grams of PVA in one liter of deionized water. Allow the solution to fully dissolve on a stir plate overnight. The next day, pour the solution into a 50 milliliter einor tube.

Centrifuge the tube at 197 times gravity for 10 minutes. Then filter with a 0.22 micrometer sterile filter to remove any residual non dissolved PVA to fabricate BSA loaded polylactic co glycolic acid or P-L-A-G-A nanoparticles dissolve 180 milligrams of uncapped 85 to 15 P-L-A-G-A in six milliliters of methylene chloride in a glass scintillation vial vortex. The solution for two minutes dissolve the desired payload in this case 15 milligrams of BSA in 1.5 milliliters of PBS.

Add the P-B-S-B-S-A solution in two parts of the P-L-A-G-A methylene chloride solution with intermittent vortex in place, the solution on ice for five minutes and sonicate with a micro tip sonic Probe at 45 watts for 120 seconds. Add the methylene chloride, P-L-A-G-A, payload PBS solution to 24 milliliters of 2%PVA solution into portions with intermediate vortexing. Next, place a solution on ice for five minutes.

Then sonicate again for 120 seconds. Place the nanoparticle emulsion on a stir plate in a fume hood for 12 hours to allow evaporation of the methylene chloride and nanoparticle stabilization following evaporation. Transfer the solution to ultracentrifugation tubes.

Centrifuge the suspension for 60 minutes at 80, 000 times. Gravity at four degrees Celsius twice. To remove residual PVA from the nanoparticles Resus, suspend the pellet in deionized water between spins after the second centrifugation.Resus.

Suspend the pellet in eight milliliters of deionized water and centrifuge for 10 minutes at 197 times. Gravity at four degrees Celsius. To isolate the 100 nanometers of population of nanoparticles, transfer the SUP agent to a new tube and place the sample in a minus 80 degrees Celsius freezer.

After the sample is completely frozen, lyophilize it for 48 hours. Store the lyophilize particles in a desiccate at minus 80 degrees Celsius until time of use. Begin by first performing a toe pinch to verify the depth of anesthesia of a ketamine hydrochloride xylazine injected mouse harboring a glioma tumor.

Next, cannulate the tail vein and set the IV to continuously infuse the appropriate microbubble solution. To initiate therapeutic low frequency treatments, set the ultrasound to initiate one burst pulse sequences. The one burst pulsing sequence consists of 100 consecutive, one megahertz sinusoids each of one volt peak tope amplitude from a waveform generator.

Amplify the waveform signal with a 55 decibel radio frequency with a power amplifier. Place the ultrasound probe above the flank tumor and apply low frequency ultrasound to the tumor for 60 minutes to destruct circulating microbubbles or microbubble nanoparticle composite agents to examine nanoparticle biodistribution following treatments. Imaging of the tumor area is performed using a fluorescence mediated tomography system prior to imaging place an anesthetized mouse in the work area, shave the imaging site flank to liver to removal hair, which will interfere with fluorescent imaging.

Next place the mouse on the imaging cartridge of the fluorescent mediated tomography system using white light and fluorescent modes. Acquire reflectance images. Then carry out fluorescent tomographic imaging in the VT six 80 channel.

The fluorescence mediated tomography system will automatically process the data using a normalized born forward equation to calculate three dimensional fluorochrome concentration distribution. This will generate a 3D image of the fluorochrome distribution within the scanned region. Following the reconstruction, draw a 3D region of interest to define the volume of interest.

Here in vivo, ultrasound of microbubble injected mice is used to mechanically ablate tumor tissue. Begin my first performing a toe pinch to verify the depth of anesthesia of a ketamine hydrochloride xylazine injected mouse harboring a glioma tumor to perform tumor perfusion measurements. Cannulate the tail vein to administer microbubbles, then infuse a lipid microbubble solution at a rate of 15 microliters per minute.

With a continuous infusion pump, apply a water-based ultrasound gel to the flank region of the mouse. Then using the 15 LH probe, scan the tumor in B mode to obtain the best imaging plane. Next, switch to CPS capture mode.

Acquire a continuous video capturing microbubble signal intensity for five seconds. Then initiate a high amplitude burst pulse using the microbubble destruction function at a frame rate of 13 hertz and continue imaging for 20 seconds following the high amplitude burst pulse. Repeat measurements in four imaging planes 10 minutes following tumor perfusion.

Add acoustic gel to the tumor. Then position a 0.5 inch diameter, one megahertz unfocused transducer over the flank tumor. Insert a needle thermocouple probe two centimeters into the tumor.

Record the temperature measurements every five minutes throughout the procedure. Set the IV to continuously infuse albumin micro bubbles. Next to perform ablative treatment, input the pulsing sequence to insensate with the five burst medium pulsing sequence, which consists of 5, 001 megahertz.

Sinusoids each of one volt peak to peak amplitude. Then turn on the signal generator, amplifier and oscilloscope. 10 minutes after the therapeutic treatment, repeat the tumor perfusion measurements as before, following either targeted drug delivery or mechanical ablation.

Use digital calipers to take measurements of the tumor length, width, and height for each measurement. Calculate tumor volumes using an ellipsoid approximation. V equals one, six pi times A, B, C, where A, B, and C are the maximum diameters of the tumor measured in three orthogonal planes.

Repeat this process each day for seven days following the treatment to monitor the effectiveness of treatment. To prepare tissue samples to evaluate the effectiveness of treatments on tumor progression. Cannulate the left ventricle of a euthanized mouse, exsanguinate the blood with 10 milliliters of 2%heparinized tris calcium chloride buffer, followed by 10 milliliters of tris calcium chloride buffer perfusion.

Fixed the tissue with an infusion of 4%paraldehyde in PBS at four degrees Celsius for 10 minutes. At 100 of mercury, allow the sample to fix for 60 minutes. Remove the skin above the flank tumor and excise the entire tumor using a surgical blade, then embed the tumor para film and cut it into five micron sections.

Tumor sections can then be stained to evaluate tumor necrosis, apoptosis, and proliferation. If this protocol is performed properly, nanoparticles will be spherical in shape as determined by scanning electron microscopy and have a Gaussian distribution around 100 nanometers as determined by light scattering techniques as shown here. Ultrasound medicated microbubble destruction results in enhanced nanoparticle delivery to tumor tissues.

The MNCA delivery technique results in heightened nanoparticle delivery immediately following treatments. Prolonged ultrasonic microbubble destruction at relatively low duty cycles, results in regression of tumor growth here, changes in tumor perfusion before and after ablative treatment are shown. In addition, we have demonstrated that prolonged microbubble intonation results in apoptosis, tissue necrosis, and a decrease in the rate of glioma growth.

While attempting this procedure, it's important to keep microbubbles in an airtight container with Okta flora propane above the aqueous phase. This technique has the potential to advance the adaptation of transcranial high intensity focused ultrasound in the clinic by providing a method for the non-thermal ablation of tumors at reduced intensity levels. In addition, this technique has the potential to increase the therapeutic index of the drug by localizing its pharmacological activity to the site or organ of interest.

In turn, this will decrease side effects, allowing for decreased dosages and or frequency of administration that is required to realize the desired therapeutic concentration.