Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

This protocol can be used to characterize the response of fluorescently labeled microbubbles that are designed for ultrasound-triggered drug delivery applications which includes the activation mechanisms and bioeffects. The key lies in the combination of imaging techniques which allows us to unravel the multi-scale problem of ultrasound-triggered drug delivery with bubbles, both at different spatial scales and at different time scales. For single bubble imaging by Brightfield microscopy, place a 19 gauge venting needle and use a one milliliter syringe equipped with a 19 gauge needle to remove a small amount of the microbubble suspension from the glass vial into a small tube for easier pipetting in the next step.

Using a pipette, dilute the microbubble solution in filtered phosphate buffered saline. Use a 10 milliliter syringe to inject the sample into one outlet of the sample holder until the holder is full without creating air bubbles, injecting more of the sample if necessary. Close both valves of the sample holder and place the sample holder perpendicular to the optical axis of the microscope.

Before sample analysis, set the desired ultrasound driving frequency and acoustic pressure on the arbitrary waveform generator and starting with the field of view at one corner of the sample holder, use the XYZ stage to move the holder to locate single microbubbles in the field of view of the microscope, then attach an optical fiber connected to a water bath to a strobe light and start the recording. Repeat the imaging as many times as desired per ultrasound setting, moving the sample holder at least two millimeters to a field of view containing unsonicated microbubbles for each analysis. For fluorescence microscopic imaging of the microbubbles, after diluting the microbubble solution in phosphate buffered saline as demonstrated, set the desired ultrasound driving frequency and acoustic pressure on the arbitrary waveform generator and set the trigger delay for the laser on the pulse delay generator for fluorescence excitation of the nanoparticles from the microbubbles, then use the XYZ stage to move the sample holder to locate single microbubbles and bring them into the focus of the objective, then trigger the recording.

Repeat the imaging as desired by altering the ultrasound settings and moving the sample holder to the new field of view as demonstrated. For imaging by intravital microscopy, first position a heated animal holder on the XY positioning stage between the wave guide and the objective and add coupling gel to the wave guide. Insert a tail vein catheter into the tail vein of an anesthetized tumor-bearing mouse and place the mouse fitted with a window chamber into the heated holder.

Add a water droplet to the coverslip. To visualize the tumor tissue vasculature, intravenously inject 30 microliters of four milligrams per milliliter fluorescently labeled 2 megadalton dextran into the tail vein catheter and use the XY translation stage to move the mouse until a field of view with suitable blood vessels can be located. Adjust the frame rate, field of view, and length of the recording according to the parameters of the experiment and record baseline images of the vessels.

When the baseline images have been acquired, set the ultrasound driving frequency, pulse length, and acoustic pressure amplitude on the arbitrary waveform generator and intravenously inject 50 microliters of microbubble sample into the tail vein. Then image the vasculature as demonstrated. Analysis of the microbubbles by confocal fluorescence microscopy reveals a non-uniform particle distribution of the microbubble shell.

The overall structure of the microbubbles can be further visualized by scanning electron microscopy. Analysis of the radial dynamics and phenomenological behavior of insonified microbubble by Brightfield microscopy allows the valuation of the relative change in the microbubble radius over time. Here, an image sequence of a typical successful delivery of fluorescently labeled nanoparticles is shown.

The nanoparticles embedded in the microbubble shell can be observed to fluoresce when the laser light reaches the bubble. As observed in this unsuccessful delivery, however, the fluorescent nanoparticles light upon the shell of the microbubble, which remains intact during the ultrasound exposure. Intravital multi-photon imaging can be used to determine the spatial and temporal extravasation of the nanoparticles during ultrasound exposure, which can be beneficial for understanding and optimizing the mechanisms underlying ultrasound-mediated nanoparticle delivery.

With the perfect alignment of the optical and acoustical pathways at all length and time scales, a comprehensive insight into ultrasound-triggered drug delivery is provided. The answers delivered by our multi-scale experiments will now be translated to clinical practice. The results will provide valuable insight for a range of therapeutic applications, including cancer therapy.