Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

Summary

This protocol outlines the fabrication of lipid microbubbles and a compatible one-pot microbubble radiolabeling method with purification-free >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.

Abstract

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 >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.

Introduction

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 cores^1,2. 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 stimul....

Protocol

1. Preparations of reagents

1. Prepare ammonium acetate buffer (0.1 M, pH 5.5)
   1. Using an analytical balance, weigh 770.8 mg of ammonium acetate onto a weigh paper. Transfer the weighed amount to a clean 250 mL glass beaker.
   2. Add 90 mL of double distilled water (ddH₂O), measured via a graduated pipette, to the beaker. Add a stir bar and place the beaker on a magnetic stir plate to dissolve the ammonium acetate. Stir at a speed that creates a slight vortex but witho.......

Discussion

The current lipid microbubble radiolabeling protocol achieves >95% radiochemical purity, >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.......

Materials

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