The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to αvβ3, a known tumor neovasculature biomarker.
Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant αvβ3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.
Molecular imaging with targeted microbubbles has been in research and testing for more than two decades. The general concept is straightforward: gas-filled microbubbles that possess selective affinity to the molecular biomarker specific to vascular endothelium in the area of disease are injected intravenously. These particles circulate and accumulate in the target (e.g., tumor neovasculature or the area of ischemic inflammatory injury). Adherent microbubbles are then detected by contrast ultrasound imaging. Early concept research efforts from last century1,2 are now gradually progressing towards clinical adoption: they have reached medium-scale clinical trial stage just several years ago3,4. The purpose of this manuscript is to provide the detailed explanation on the preparation and characterization of such targeted microbubbles, based on two published examples1,5.
The procedure for the preparation of peptide-PEG-phospholipid, a crucial component for the formulation of these targeted microbubbles, is supplemented with the description of reagent quality control, as required for the successful completion of the reaction. Unfortunately, some active ester lipid reagent suppliers provide material that is hydrolyzed on arrival and therefore is unable to participate in the formation of amide bond. The information on how much of the lipid material is transferred onto the microbubble shell from the aqueous medium during microbubble preparation is provided, as well as the technique to obtain this information.
It is important to prepare microbubbles with a relatively narrow particle size distribution: the co-presence of large microbubbles in the injectable media for intravascular in vivo testing may lead to clogging of microvasculature; nonspecific accumulation of microbubbles that bypass lung shunts might cause nonspecific false-positive tissue enhancement6, which is avoided by removing microbubbles of larger sizes. Therefore, a simple procedure to achieve particle size selection is presented, supplemented by the description of a method to assess particle concentration and size distribution with a particle counter.
The first test protocol for microbubble targeting assessment, as presented below, describes a purely model system, with biotinylated microbubbles targeted to streptavidin-coated surface1. The second protocol is based on a manuscript describing simplified preparation of peptide-targeted microbubbles, decorated with a cyclic RGD peptide that possesses specific affinity towards αvβ3, a molecular biomarker of tumor neovasculature5. Microbubbles decorated with this cyclo[Arg-Gly-Asp-D-Phe-Lys], i.e., c(RGDfK) peptide by the presented technique have been shown to target tumor neovasculature and achieve ultrasound molecular imaging in a murine tumor model.
The importance of a simple technique for the preparation of ligand-decorated microbubbles is evident. The use of amalgamation technique for microbubble preparation, as pioneered by Unger et al.,16 may serve this purpose for a number of reasons. Manufacturing of microbubbles by amalgamator is easy to perform. A small-footprint desktop single-phase 120 V unit is available and inexpensive. The procedure is quick (45 seconds) and efficient: 1 mL of microbubble dispersion in an aqueous medium is prepared at once. It contains billions of particles per mL, more than sufficient for research studies. Manufacturing occurs in a sealed vial with perfluorinated gas headspace. If necessary, vial contents will remain sterile from the time of aseptic filling, during manufacturing (amalgamation), and until use. This makes the approach relevant for clinical use, as it does not require elaborate preparations in a dedicated sterile environment in clinic.
The procedure is based on self-assembly: during mixing, as high shear is applied to gas-water interface within the moving vial, small gas fragments are formed, which assume spherical shape due to action of surface tension. PG, as a cosurfactant, present in the medium at high concentration, reduces surface tension and energy required to generate gas-water interface during shear. Next, more “classical” surfactants, such as PEG-lipids and phospholipids, which are present at much lower concentrations, get to the interface, most likely displacing PG and establishing a monomolecular layer at the bubble surface. This shell is reasonably stable; it is likely due to a combination of a “solid” lipid (DSPC phase transition temperature is 56 °C, so it is not prone to inter-membrane fusion) and an extended PEG brush coat that surrounds the microbubbles and inhibits direct monolayer contact of neighboring bubbles. One can speculate that the presence of a high concentration of PG in the media may lower the microbubble shell stability. In its absence, microbubbles are stable in the sealed vials under fluorocarbon atmosphere for many months, with only moderate fusion between the bubbles. For clinical use, with a small amalgamator device at the bedside, the interval between microbubble preparation and use can be short, minutes or hours. With PG present in the media, microbubble concentration does not show a significant drop, at least for several hours of refrigerated storage.
An added advantage of the described procedure (assisted by the use of PG cosurfactant in the bubble preparation medium) is high efficacy (>85%) of the lipid transfer to the shell, whereas the traditional sonication provides ~20% efficacy5 and modern microfluidic methods even lower17. High level of transfer efficiency is important not just because the waste of lipid material and expensive ligand is reduced, but because the amount of bubble-free ligand co-present in the media is minimized as well. Then the free ligand may not have an opportunity to block the biomarker target receptor to which the microbubbles are expected to bind via ligand on their surface. The general amount of the biomarker receptor on the target vasculature is often quite high, so this might not be of utmost importance. From the available patent literature18 one might suggest that at least 50% of lipid shell material and targeting ligand in the microbubble formulations in clinical testing may be associated with the bubble shell. This can be generally compared with radiolabeled antibodies or peptides that are widely used in nuclear medicine receptor imaging studies: most of those targeting ligand molecules actually do not carry “hot” radioisotope even for the highest specific activity reported19, whereas for targeted microbubbles, the shell material in this study (including ligand-lipid) is mostly attached to microbubbles.
Selective adhesion of targeted microbubbles prepared by this technique in vitro was demonstrated, in two sets of targeting models: static adhesion, and a flow chamber targeting experiment. In a static assay, targeted microbubbles adhered to the target receptor layer tightly and were not dislodged with buffer rinse, unlike in a control setting, where microbubbles were removed from the surface even with a gentle rinse. Likewise, in a flow-through test, performed in a parallel plate flow chamber, biotinylated bubbles demonstrated statistically significant and superb adhesion to streptavidin layer on a polystyrene dish, when compared with the control albumin-only surface. Peptide c(RGDfK)-decorated microbubbles selectively adhered to αvβ3-coated surface, both in the static adhesion assay, and in a parallel plate flow chamber.
The following issues can be considered as the limitations of the described protocol. First, the procedure does not account for the submicron particles. The instrument that was used in the study was not set up to detect nanobubbles (i.e., particles under 1 μm in diameter). These particles might have been present in the formulation. Although their acoustic backscatter signal is generally known to be low, and they were not observed in this study by microscopy, the presence of nanobubbles should still be considered. The second significant issue is the size heterogeneity of the microbubbles. Despite the removal of larger particles, the size of the resulting bubbles is far from uniform. This should be a consideration and justification for further research in the area of microbubble formulation.
In conclusion, the narrative given in this manuscript should provide sufficient level of technical detail to manufacture targeted microbubbles quickly and easily. The steps to perform additional purification (if desirable), adjusting the size and/or assessing the small amount of the shell material that remains in the aqueous medium are provided. The detailed analytical tools for the assessment of the microbubble parameters, such as size distribution and concentration, and in vitro ability of ligand-decorated microbubbles to adhere to target receptors are described.
The authors have nothing to disclose.
A.L. Klibanov acknowledges support in part via NIH R01EB023055, awarded by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health, a subcontract to University of Virginia via NIH R01NS076726, awarded to UCSF by National Institute of Neurological Disorders and Stroke of the National Institutes of Health, and a subcontract to University of Virginia via NIH grant R44HL139241, awarded to SoundPipe Therapeutics by National Heart, Lung, and Blood Institute. The content of this publication is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.
Amalgamator | Lantheus, Billerica, MA. | Vialmix | ESPE Capmix, Wig-L-Bug or another amalgamator capable of 4300 rpm can be used. |
biotin-PEG3400-DSPE | Laysan Bio, Arab, AL. | Biotin-PEG-DSPE-3400 | |
Bovine Serum Albumin (BSA) | Fisher Scientific, Waltham, MA | BP1600-100 or similar | |
Ca-Mg-free PBS | Fisher Scientific, Waltham, MA | 14190-144 | |
Centrifuge with a bucket rotor | IEC/Thermo, Fisher Scientific, Waltham, MA. | NH-SII | Any centrifuge with a bucket rotor |
chloroform | Fisher Scientific, Waltham, MA | C297-4 | |
cyclic (RGDfK) peptide | AnaSpec, Fremont, CA | AS-61111 | |
Decafluorobutane | F2 Chemicals, Preston UK | CAS 355-25-9 | |
DiI | Sigma-Aldrich, St. Louis, MO. | 468495-100MG | |
DIPEA | Sigma-Aldrich, St. Louis, MO. | 387649 | |
Disposable UV cuvette, 1.5 ml | BrandTech, Essex, CT. | 759150 | |
DMSO | Sigma-Aldrich, St. Louis, MO. | 276855 | |
Dry block heater, high temperature | Techne Cole Palmer, Staffordshire UK | DB-3A | |
DSPC | Lipoid, Ludwigshafen, Germany | LIPOID PC 18:0/18:0 | |
Fluorescence microplate reader | Molecular Devices, San Jose, CA. | Spectramax Gemini XS | No longer available, superceded by Gemini XPS; any fluorescence plate reader with red dye detection capability will work |
Microscope with fluorescence epi-illumination. | Leica | Laborlux 11 | No longer available; any fluorescence microscope is sufficient; high-sensitivity video camera is required for image stream collection |
NHS-PEG3400-DSPE | NOF-America, White Plains, NY. | SUNBRIGHT DSPE-034GS | Some of the alternative manufacturers provide material that is mostly, or completely, hydrolyzed on arrival |
Ninhydrin spray for TLC plates | BVDA, Haarlem, The Netherlands | AS-72177 | |
Normal saline irrigation solution (0.9% NaCl) | Baxter, Deerfield IL. | 2F7124 | |
Parallel plate flow chamber, for 35mm Corning Petri Dish | Glycotech, Gaithersburg, MD. | 31-001 | May only work with Corning Petri dishes, but not necessarily with other makers, due to different dimensions |
Particle sizing system | Beckman Coulter, Hialeah, FL | Multisizer 3 | No longer available, superceded by Multisizer 4, with similar electrozone sensing principle. Alternatively, optical methods, e.g., Accusizer, can be used. |
PEG 6000 monostearate | Kessco Stepan, Joliet, IL. | CAS 9004-99-3 | |
Petri Dishes, 35 mm diameter, 10 mm tall | Corning, Corning, NY. | 430165 | |
Plastic coverslips, 22x22mm | Cardinal Health, McGaw Park, IL. | M6100 | |
Propylene glycol | Sigma-Aldrich, St. Louis, MO. | P4347 | |
Recombinant murine alphavbeta3, carrier-free | R&D Systems, Minneaposis, MN. | 7889-AV-050 | |
Rubber stoppers, 13mm | Kimble-Chase, Vineland, NJ | W017900 | |
Serum vials, 2 ml, 13mm | Kimble-Chase, Vineland, NJ | 223683 | |
Silica TLC Plates, F254 | Analtech, Newark, DE | P02521 | |
Sodium tetraborate | Sigma-Aldrich, St. Louis, MO. | S9640 | |
Streptavidin | AnaSpec, Fremont, CA | AS-72177 | |
Syringe pump, infuse/withdraw option | Harvard Apparatus, Holliston, MA | PHD2000, 70-2001 | |
Ultrasound imaging system with contrast-specific mode. | Siemens/Acuson, Mountain View CA | Sequoia c512, 15L8 probe | Old generation Sequoia is out of production for more than a decade. Available as used equipment. CPS mode has to be unlocked for the 15L8 transducer. |
UV Spectrophotometer | Beckman, Brea, CA. | DU640 | No longer available, may be replaced with any 260 nm ultraviolet-capable unit |
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