Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

Microbubbles are gas-filled ultrasound-susceptible particles with unique cancer imaging and therapy capabilities. Their evolving utilities can be strategically advanced by building application-driven traceable microbubbles. We aim to facilitate this goal by developing a lipid microbubble fabrication protocol that allows for customizable generation of unlabeled, radiolabeled, fluorescent and multimodal microbubbles.

Achieving efficient and stable microbubble radio labeling is a non-trivial challenge. Lipid microbubbles are fragile and thus subjecting them to heat, time and purification steps in post fabrication radio labeling approaches can perturb their physical chemical properties and stability. On the other hand, prefabrication radio labeling requires specialized equipment and increased radiation exposure.

We overcome these challenges by developing a new hybrid protocol in which radioisotope chelation occurs prior to microbubble activation, but after lipid suspension formation. This one-pot approach for the first time facilitates high efficiency, purification-free microbubble radio labeling that preserves particle physical chemical properties. A key additional advantage is the protocols versatility. It can be incorporated during ground up custom microbubble fabrication and adapted to preformed microbubble lipid suspensions such as those available commercially. It can be successfully applied across diverse microbubble lipid and chelator compositions, and can accordingly generate tailored radioactive, fluorescent or trimodal microbubbles.

Together, these properties enable multimodal representative tracing of microbubble lipid shells that can advance mechanistic insights and support the advancement of microbubble-focused ultrasound therapies across many domains, including microbubble pharmacokinetic study, multimodal imaging, treatment planning, and enabling synergistic therapies.

[Narrator] To begin using a micro pipette, add one milliliter of AA-PGG solution along the edges of the microbubble lipid film of choice in a vial, avoiding bubble formation. Partially cover the vial opening with the cap, leaving enough space to insert the perfluoropentane or PFP line. Flow PFP into the headspace of the vial for 20 seconds above the liquid and cap the vial. Submerge the bottom half of the vial into a 70 to 80 degrees Celsius water bath for one minute. Then sonicate the vial for 30 seconds in 69 degrees Celsius baths sonicator. After sonication, wipe the vial and allow it to cool to room temperature. Refill the headspace of the vial with PFP gas. Then cap the vial and seal the edges with Parafilm. To set up a 60 degree Celsius water bath in a crystallizing dish, place a magnetic stir bar in the dish and position it on a temperature controlled hot stir plate. Insert a thermal probe into the water to monitor the temperature and adjust the stirring speed to create a weak but visible funnel. Using forceps, transfer a sealed vial containing copper 64 in the form of cuperic chloride in 0.1 normal hydrochloric acid to a dose calibrator. Record the copper 64 activity and the time. Using forceps, remove the vial from the dose calibrator and place it in a leaded container. To calculate the value in megabecquerels per milliliter, divide the activity noted on the dose calibrator by the volume of copper 64. Uncap the lipid suspension and secure it in a vial holder. Then uncap the copper 64 vial, handling the vial with forceps. Using a micropipet transfer a volume of copper 64 solution corresponding to 40 to 250 megabecquerels of activity into the lipid suspension. Using flat rubber tipped forceps, rotate the radioactive lipid suspension up and down at least five times to mix the copper 64 into the suspension. While the vial is right side up, gently flick the cap while stabilizing the vial. Carefully, partially uncap the vial and insert a needle equipped PFP line. Fill the vial headspace with PFP for 20 seconds. Then cap and seal the vial with parafilm. Measure the vial activity using a dose calibrator and record the time. Next, place the vial in a foam vial holder so that the bottom half of the vial is exposed to heat. Submerge the holder in the 60 degrees Celsius stirring water bath and heat for one hour. Meanwhile, to prepare the iTLC strips, cut glass microfiber chromatography paper into strips. Heat the strips in an 80 degrees Celsius glass drying oven to activate. After one hour, remove the vial from the water bath and wipe its edges with tissue paper. Rotate the vial up and down using rubber tipped forceps. While the vial is in an upright position, flick the cap while stabilizing the vial. Remove the parafilm and wipe around the cap to remove any trapped water. Spot one to two microliters of the lipid suspension one centimeter from the bottom center of an activated iTLC strip. Allow the spot to dry. Using a micropipette, add 200 microliters of iTLC eluent into the bottom of a 10 milliliter test tube. House the tube in a lead container. Add the spotted iTLC strip to the tube and allow it to develop until the eluent reaches approximately one centimeter from the top of the strip. Next, using forceps, remove the developed strip and hold it vertically. Cut the strip into thirds over gamma counter compatible five milliliter plastic tubes. Insert push caps into the tubes. Measure the strip containing tubes and an empty control tube using a gamma counter to detect copper 64 activity. Record the counts per minute and subtract the control tube activity from the other readings to correct for background radiation. Then calculate the radio chemical purity based on the corrected counts per minute. Next, uncap the radio labeled lipid suspension vial and add 8.89 microliters of one normal sodium hydroxide into the suspension. After capping and rotating the vial, tap the vial gently to remove trapped suspension in the cap space and fill the head space with PFP gas. Place the vial on a mechanical shaker and activate it for 45 seconds at 4,530 RPM to generate a milky microbubble suspension. Upon passive cooling to room temperature, gently invert the vial to resuspend the microbubble suspension. Set the vial on a flat surface and wait for two minutes. Meanwhile, equip a one milliliter plastic syringe with an 18 gauge needle and vent the syringe by aspirating and plunging air in and out. After two minutes, uncap the vial quickly and draw 400 to 550 microliters of the suspension from the bottom of the vial. Carefully wipe the edges of the needle and transfer the microbubble suspension into a micro centrifuge tube. After capping the tube, measure the activity of the final microbubble product on a dose calibrator and note the time. Divide the activity value by the volume decanted to obtain the activity in megabecquerels per milliliter. To begin, add 10 to 200 microliters of the microbubble suspension to a 0.5 milliliter, 30,000 molecular weight cutoff centrifuge filter unit in a compatible micro centrifuge tube. Centrifuge the suspension at 12,000 G for 10 minutes at room temperature. Next, use scissors to cut the connection between the micro centrifuge tube and its cap. Place the cap in a 20 milliliter scintillation vial labeled CAPS. Transfer the filter unit to a new micro centrifuge tube labeled TUBE 2. Place the first micro centrifuge tube containing infranatant into a 20 milliliter scintillation vial labeled TUBE 1. Add 200 microliters of double distilled water to the filter unit in TUBE 2. Centrifuge the filter unit again at 12,000 G for 10 minutes. After the last filter unit transfer, add 200 microliters of distilled water to the filter unit in TUBE 3 and centrifuge. After centrifugation, cut the cap of the tube, adding it to the cap scintillation vial, and transfer the filter unit to a new 20 milliliter scintillation vial labeled UNIT. Place TUBE 3 in a new 20 milliliter glass scintillation vial. Cap the five scintillation vials and prepare one empty and capped scintillation vial as a blank control. Measure the six scintillation vials on a gamma counter to detect copper 64 activity. Subtract the blank control from the other vial measurements. Calculate the radio labeling or chelation efficiency.