Name: Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers (Video) | JoVE Journal
Uploaded: 2025-01-24T14:00:00.000+00:00
Duration: 10 min 40 s
Description: Learn to fabricate lipid microbubbles and apply a one-pot radiolabeling method achieving >95% efficiency without purification. This protocol preserves physicochemical properties for cancer imaging applications.

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5 minutes

We thank Deborah Scollard and Teesha Komal (University Health Network Spatio-Temporal Targeting and Amplification of Radiation Response (STTARR) program, Toronto, Ontario) for their technical services and guidance. We also thank Mark Zheng and Dr. Alex Dhaliwal for their technical assistance during confocal microscopy and the Advanced Optical Microscopy Facility (Toronto, Ontario) for providing associated equipment. We acknowledge our funding sources: the Canadian Institutes of Health Research, the Terry Fox Research Institute, the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, the Princess Margaret Cancer Foundation, Canada Research Chairs Program, the McLaughlin Centre, the Vanier Scholarship Program, the Ontario Graduate Student Scholarship Program, Prostate Cancer Canada, and the Peterborough K. M. Hunter Charitable Foundation.

The authors report no conflicts of interest.

The current lipid microbubble radiolabeling protocol achieves &#62;95% radiochemical purity, &#62;95% chelation efficiency, and retention of microbubble physicochemical properties without necessitating any post-labeling purification. These accomplishments represent advancements previously unattained for existing labeling protocols. Lack of purification steps allows quicker use of radioisotopes (in this case, copper-64), and thus, reduction of inefficient activity loss from radioactive decay. The resulting retention of mi...

Microbubbles are micron-sized supramolecular theranostic agents with a gas core stabilized by a protein, polymer, or, in most cases, a lipid shell (Figure 1A). When injected into the bloodstream, microbubbles maintain gas/liquid interfaces that are detectable by ultrasound for minutes-long timeframes prior to the dissolution of their gas cores1,2. Consequently, the first clinical use of microbubbles was as real-time ultrasound imaging contrast agents3. The invention of therapeutic focused ultrasound (FUS) expanded microbubble clinical utilities. When stimulated by low-frequency FUS, microbubbles oscillate and generate targeted, tunable mechanical forces ranging from transient vascular permeabilization to focal tissue ablation4,5. As a result, over the last 20 years, microbubble-FUS has been explored for blood-brain barrier (BBB) opening, tumor (e.g., pancreatic, brain, and liver metastatic cancer) drug and imaging probe delivery, neurodegenerative disease therapy and cancer ablation6,7,8,9,10,11.
The theranostic arsenal of microbubbles continues to advance in new and exciting directions. Conventional microbubble-FUS delivery applications rely on the co-administration of therapeutic or imaging cargo alongside commercial microbubbles. There is growing interest in enhancing microbubble-FUS delivery capabilities by understanding microbubble shell/biological interactions, exploring custom-made non-commercial microbubble formulations, and generating all-in-one theranostic microbubbles with cargo loaded directly onto the microbubble shell12,13,14. In fact, approximately 40% of lipid microbubble drug delivery studies make use of such shell-loaded microbubbles15. Beyond imaging and drug delivery, microbubble-FUS has also shown promise in enhancing cancer radiotherapy16, and activating antineoplastic effects of otherwise benign shell-loaded agents through sonodynamic therapy17,18.
These conventional and expanded directions in microbubble cancer applications can be more strategically advanced by labeling microbubble shells with radioactive tracers. In the realm of all-in-one cargo-loaded microbubbles, such radiolabeling 1) facilitates gold-standard, quantitative assessment of the on and off-target biodistribution of these loaded microbubble shells, 2) derives pharmacokinetic structure-activity relationships that inform optimal selection of microbubble compositions to maximize on-target delivery, and 3) guides strategic and appropriate image-guided application and treatment planning (e.g., types of tissue targets, dosimetry, drug selection to mitigate off-target safety concerns, utility compared to conventional co-treatment paradigms) of all-in-one cargo-loaded systems15,19. At a preclinical stage, such an understanding of microbubble shell fate can also illuminate broader microbubble-FUS mechanisms of action. For example, lipid transfer from microbubble shells to target cells has been shown to influence FUS-enabled sonoporation12,20. Understanding and optimizing such transfer can thus inform preclinical and clinical microbubble-FUS therapies in which sonoporation is implicated (in vitro transfection, drug delivery, tumor ablation, radiation sensitization, and sonodynamic therapy20,21,22,23,24,25). Dual ultrasound and radioimaging facilities would also enable FUS vessel opening and treatment monitoring (e.g., BBB opening kinetics) from a single agent rather than conventional dual agent designs26. In the same vein, lipid microbubble radiolabeling could serve as an all-in-one single-agent microbubble-FUS/radiotherapy alternative to microbubble-FUS + radiopharmaceutical co-delivery platforms27.
The fragility of microbubbles is an untrivial challenge to such labeling. All existing radiolabeling strategies are limited by purification methodologies known to perturb microbubble stability and size, while some also feature ineffective and unstable radiolabeling28,29,30,31,32. Purification requirements also lead to lengthier protocols. Combined with the use of short-lived radioisotopes (e.g., 18F t1/2 1.8 h,28,29 99mTc t1/2 6 h,32 68Ga t1/2 1 h31), this creates inefficiencies related to radioisotope decay and confines radioimaging and treatment planning timeframes. Collectively, these limitations risk the acquisition of shortened and unrepresentative radioimaging, inaccurate pharmacokinetic data, and inefficient tumor radioisotope delivery.
In this report, these limitations are overcome by leveraging the strong and stable metal chelation capabilities of porphyrin. Porphyrins are organic, heterocyclic macromolecules with a highly conjugated planar ring and a central coordination site that can accommodate a variety of metals. This includes longer-lived radioisotopes such as copper-64 (t1/2 12.7 h), a radiopharmaceutical with positron emission tomography (PET), and γ-counting feasibilities33. When conjugated to a lipid backbone, porphyrins can be readily incorporated into supramolecular structures and subsequently labeled with copper-64 with speed, high chelation efficiency, and serum stability, while maintaining the properties of the parent unlabeled particles33,34. Furthermore, porphyrins are fluorescently active with modular self-quenching in nano and microparticles that is restored upon particle disruption; a complementary readout to PET and γ-counting that facilitates both bulk and microscopic shell fate analysis (Figure 1A)15.
By using porphyrin-lipid as a chelator, these properties were exploited to generate a new one-pot, purification-free microbubble radiolabeling methodology (Figure 1B,C) that overcomes limitations associated with existing microbubble radiolabeling methods. This protocol achieves &gt;95% copper-64 chelation efficiency, does not require post-labeling purification, and preserves microbubble physicochemical properties. It can be integrated easily into the "ground-up" fabrication of lipid microbubbles prior to their activation (Figure 1B). It is versatile and can be applied successfully across custom and commercial microbubble formulations with differing acyl lipid chain length (C16 to C22), charge (neutral and anionic), and porphyrin-lipid compositions (1 mol%, 10 mol%, 30 mol%), generating microbubbles with both radio and fluorescence activity. Its adaptability can also extend beyond porphyrin. The one-pot protocol can be modified to use alternative commercially available chelators (e.g., diethylenetriamine pentaacetate (DTPA)-lipid) and fluorophores (e.g., DiI). It can also be modified to label pre-made microbubble formulations through a "spiking" approach. Accordingly, this method enables the production of tailored, traceable (radio, fluorescent, or dual radio/fluorescent active) microbubbles useful for advancing mechanistic, imaging and therapeutic microbubble-FUS applications. The protocol below outlines the fabrication of lipid microbubbles, application of the one-pot radiolabeling protocol, requisite radiolabeling and physicochemical property characterization, and potential modifications.
<img alt="Porphyrin-lipid microbubble formation diagram; fluorescence, ultrasound, radiolabeling process setup." class="xfigimg" src="/files/ftp_upload/67431/67431fig01v4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Microbubble fabrication and radiolabeling protocol. (A) Porphyrin-lipid, in the form of pyropheophorbide-a-lipid, serves as a multimodal chelator within this protocol. As a monomer chelated to copper-64 (i), it has PET and imaging capabilities. Its fluorescence is quenched in particle form (microbubbles (ii) and their post-dissolution nanoprogeny (iii)) and unquenched with particle disruption (iv). (B) Lipid film hydration/activation protocol described in this report to generate lipid microbubbles from the ground-up and (C) integration of one-pot radiolabeling between lipid suspension formation and microbubble activation. This figure was adapted with permission from Rajora et al.15. <a href="https://www.jove.com/files/ftp_upload/67431/67431fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>64CuCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Washington University School of Medicine, Mallinckrodt Institute of Radiology</td><td>N/A</td><td>Order in small volume (&amp;lt;10 &amp;micro;L) dissolved in 0.1 N HCl</td></tr><tr><td>Acetic acid&amp;nbsp;</td><td>Any company</td><td></td><td>&amp;ge; 95% purity</td></tr><tr><td>Aluminum foil</td><td>Any company</td><td></td><td></td></tr><tr><td>Ammonium acetate</td><td>Any company</td><td></td><td>Purity: &amp;ge; 98%</td></tr><tr><td>Balance - analytical</td><td>Any company</td><td></td><td>Able to measure down to 0.1 mg</td></tr><tr><td>Bath sonicator</td><td>Any company</td><td></td><td>Can be heated to 69 &lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>CC aperture - 30 micron</td><td>Beckman Coulter</td><td>A36391</td><td>Particle diameter range: 0.6-18 um</td></tr><tr><td>CC electrolyte</td><td>Beckman Coulter</td><td>8546719</td><td>Isoton II diluent</td></tr><tr><td>CC Software</td><td>Beckman Coulter</td><td>Multisizer 4e</td><td></td></tr><tr><td>Centrifuge filter units (0.5 mL 30,000 MWCO) with compatible microcentrifuge tubes</td><td>MilliporeSigma</td><td>UFC503096</td><td>Amicon Ultra - 0.5 mL</td></tr><tr><td>Centrifuge tubes - 15 mL with caps</td><td>Any company</td><td></td><td></td></tr><tr><td>Chloroform</td><td>Any company</td><td></td><td>Purity: &amp;ge; 99.8%&amp;nbsp;</td></tr><tr><td>Coulter counter</td><td>Beckman Coulter</td><td>B43905</td><td>Multisizer 4e Coulter Counter</td></tr><tr><td>Cover slips</td><td>VWR</td><td>48393081</td><td>VWR micro cover glass</td></tr><tr><td>CuCl2</td><td>Any company</td><td></td><td>Ensure not oxidized</td></tr><tr><td>CuCl2</td><td></td><td></td><td></td></tr><tr><td>Cuvette- quarts, 1 cm path length</td><td>Any company</td><td></td><td></td></tr><tr><td>Cuvettes - 10 mL plastic for CC measurements</td><td>Beckman Coulter</td><td>A35471</td><td>Coulter Counter Accuvette ST</td></tr><tr><td>ddH2O</td><td>Any company</td><td></td><td>Can be obtained through an ultrapure water purification system</td></tr><tr><td>DiI (1,1&#039;-Dioctadecyl-3,3,3&#039;,3&#039;-Tetramethylindocarbocyanine Perchlorate)</td><td>Any company</td><td></td><td>Powder form</td></tr><tr><td>Dose calibrator</td><td>Any company</td><td></td><td>Able to read copper-64</td></tr><tr><td>DPPA (1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt))</td><td>Avanti Polar Lipids</td><td>830855P</td><td>Powder form</td></tr><tr><td>DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)</td><td>Avanti Polar Lipids</td><td>850355P</td><td>Powder form</td></tr><tr><td>DPPE-MPEG (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt))</td><td>Avanti Polar Lipids</td><td>880200P</td><td>Powder form</td></tr><tr><td>DTPA-lipid (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (ammonium salt))</td><td>Avanti Polar Lipids</td><td>790106P</td><td>Powder form</td></tr><tr><td>EDTA (Ethylenediaminetetraacetic acid)</td><td>Any company</td><td></td><td></td></tr><tr><td>Gamma counter</td><td>Any company</td><td></td><td>Able to read copper-64</td></tr><tr><td>Gamma counting tube push caps</td><td>Globe Scientific</td><td>22-171-665</td><td>Flanged plug caps for 12 mm tubes</td></tr><tr><td>Gamma counting tubes</td><td>Sarstedt</td><td>55.1579</td><td>5 mL, 75 x 12 mm, PS</td></tr><tr><td>Glass beaker - 250 mL</td><td>Any company</td><td></td><td>Able to withstand temperatures up to 100 &lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Glass drying oven</td><td>Any company</td><td></td><td>Can be heated to 80 oC</td></tr><tr><td>Glass microliter syringes - 25, 50, 100, 1000 &amp;micro;L</td><td>Any company</td><td></td><td>Compatible with organic solvents</td></tr><tr><td>Glass scintillation vials - 20 mL</td><td>VWR</td><td>66022-081</td><td>VWR&amp;reg; Scintillation Vials, Borosilicate Glass, with Screw Caps, With pulp foil liner</td></tr><tr><td>Glass vials - 0.5 dram</td><td>VWR</td><td>66011-020</td><td>VWR Vial 1/2 dram, with black phenolic screw cap and polyvinyl-faced pulp liner</td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G7757-1L</td><td>Purity:&amp;nbsp; &amp;ge; 99.0%&amp;nbsp;</td></tr><tr><td>Graduated pipette/gun</td><td>Any company</td><td></td><td></td></tr><tr><td>Hot/stir plate</td><td></td><td></td><td>Equipped with temperature prob for automatic tempearture control</td></tr><tr><td>Hydrochloric acid - 0.1 N</td><td>Any company</td><td></td><td></td></tr><tr><td>iTLC plates</td><td>Agilent</td><td>A120B12</td><td>&amp;nbsp;iTLC-SA chromatography paper</td></tr><tr><td>Laboratory tissues</td><td>Any company</td><td></td><td></td></tr><tr><td>Media vaccuum filtration unit</td><td>Any company</td><td></td><td>0.22 micron pore size, PES membrane, 500 mL funnel capacity</td></tr><tr><td>Methanol</td><td>Any company</td><td></td><td>Purity:&amp;nbsp; &amp;ge; 99.8%, HPLC grade, meets ACS specifications</td></tr><tr><td>Microcentrifuge tubes non sterile - 1.5 mL</td><td>Any company</td><td></td><td></td></tr><tr><td>Microcentrifuge tubes sterile - 1.5 mL</td><td>Any company</td><td></td><td></td></tr><tr><td>Micropipetes - p1000, p200, p20, p10</td><td>Any company</td><td></td><td>Ensure are calibrated</td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>12-550-15</td><td>Superfrost Plus Microscope Slides Precleaned</td></tr><tr><td>Needles - 18 G</td><td></td><td></td><td>Sterile</td></tr><tr><td>Parafilm</td><td>Any company</td><td></td><td></td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>D8537-500ML</td><td>DPBS, modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture</td></tr><tr><td>PFP</td><td>FluoroMed</td><td>APF-N40HP</td><td>Purity:&amp;nbsp; &amp;ge; 99.8%</td></tr><tr><td>PFP line</td><td>Any company</td><td></td><td>1/4 inch diameter plastic hose cut about 50 cm in length</td></tr><tr><td>PFP regulator</td><td>Swagelok</td><td>SS-1RF4 and SS-4HC-1-4</td><td></td></tr><tr><td>pH meter</td><td>Any company</td><td></td><td></td></tr><tr><td>pH standards 4 and 7</td><td>Any company</td><td></td><td></td></tr><tr><td>Pipette tips for p1000, p200, p10 - non sterile</td><td>Any company</td><td></td><td></td></tr><tr><td>Pipette tips for p1000, p200, p10 - sterile</td><td>Any company</td><td></td><td></td></tr><tr><td>Plastic syringe - 1 mL</td><td>Any company</td><td></td><td>Sterile</td></tr><tr><td>Propylene glycol</td><td>BioShop</td><td>PRO888.500</td><td>Purity:&amp;nbsp; &amp;ge; 99.5%</td></tr><tr><td>Pyro-lipid</td><td>N/A</td><td></td><td>Made in-house</td></tr><tr><td>Rubber tipped forceps</td><td>Any company</td><td></td><td>Mix of fine-tipped and flat/square edges recommended</td></tr><tr><td>Scissors</td><td>Any company</td><td></td><td></td></tr><tr><td>Sodium hydroxide - 1 N</td><td>Any company</td><td></td><td></td></tr><tr><td>Sodium hydroxide - 10 N</td><td>Any company</td><td></td><td></td></tr><tr><td>Spectrofluorometer</td><td>Any company</td><td></td><td>Capable of 410 nm excitation and 600-850 nm emission</td></tr><tr><td>Spectrofluorometry software</td><td>Horiba</td><td>FluorEssence</td><td></td></tr><tr><td>Spectrometer</td><td>Any company</td><td></td><td></td></tr><tr><td>Syringe - 1 mL</td><td>Any company</td><td></td><td>Disposible, plastic, sterile</td></tr><tr><td>Syringe filters - 0.2 micron pore size</td><td>Any company</td><td></td><td>Membrane material: PES or other compatible with ammonium acetate/acetic acid and PBS</td></tr><tr><td>Test tube - 10 mL</td><td></td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Any company</td><td></td><td></td></tr><tr><td>Vacuum desicator/vacuum</td><td>Any company</td><td></td><td></td></tr><tr><td>Vialmix</td><td>Lantheus Medical Imaging</td><td>515030-0508</td><td>Referred to in protocol as a mechanical vial shaker</td></tr><tr><td>Weigh paper</td><td>Any company</td><td></td><td>To avoid losing product, cutting weigh paper into 3x3 cm squares is recommended</td></tr></tbody></table>

fabricating and labeling microbubbles with fluorescent and radioactive tracers

Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy platforms. When combined with therapeutic focused ultrasound (FUS), they can safely and locally overcome physiological barriers (e.g., blood-brain barrier), deliver drugs to otherwise inaccessible cancers (e.g., glioblastoma and pancreatic cancer), and treat neurodegenerative diseases. The therapeutic arsenal of microbubble-FUS is advancing in new directions, including synergistic combination radiotherapy, multimodal imaging, and all-in-one drug loading and delivery from microbubble shells.
Labeling microbubbles with radiotracers is key to establishing these expanded theranostic capabilities. However, existing microbubble radiolabeling strategies rely on purification methodologies known to perturb microbubble physicochemical properties, use short-lived radioisotopes, and do not always yield stable chelation. Collectively, this creates ambiguity surrounding the accuracy of microbubble radioimaging and the efficiency of tumor radioisotope delivery.
This protocol describes a new one-pot, purification-free microbubble labeling methodology that preserves microbubble physicochemical properties while achieving &#62;95% radioisotope chelation efficiency. It is versatile and can be applied successfully across custom and commercial microbubble formulations with differing acyl lipid chain length, charge, and chelator/probe (porphyrin, DTPA, DiI) composition. It can be adaptively applied during ground-up microbubble fabrication and to pre-made microbubble formulations with modular customizability of fluorescence and multimodal fluorescence/radioactive properties. Accordingly, this flexible method enables the production of tailored, traceable (radio, fluorescent, or radio/fluorescent active) multimodal microbubbles that are useful for advancing mechanistic, imaging, and therapeutic microbubble-FUS applications.

The key quantifiable results when fabricating radiolabeled microbubbles are radiochemical purity and radiolabeling efficiency. This protocol uses iTLC and a validated centrifugal procedure, respectively, to characterize each. Figure 2A shows that average radiochemical purities and efficiencies of ≥95% were achieved across commercial microbubble mimicking formulations in which the host lipid was substituted for pyro-lipid at compositions of 1 mol%, 10 mol%, or 30 mol% of the total lipid. The ...

This protocol outlines the fabrication of lipid microbubbles and a compatible one-pot microbubble radiolabeling method with purification-free &gt;95% labeling efficiency that conserves microbubble physicochemical properties. This method is effective across diverse lipid microbubble formulations and can be tailored to generate radioactive and/or fluorescent microbubbles.

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy ...

fabricating-labeling-microbubbles-with-fluorescent-radioactive

Research

JoVE Journal

Cancer Research

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Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

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Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

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Detection of Protease Activity by Fluorescent Peptide Zymography

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Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

Learn to fabricate lipid microbubbles and apply a one-pot radiolabeling method achieving >95% efficiency without purification. This protocol preserves physicochemical properties for cancer imaging applications.

Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

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