Contrast-enhanced ultrasound

Contrast-enhanced ultrasound (CEUS) is the application of ultrasound contrast agents to traditional medical sonography. Ultrasound contrast agents are gas-filled microbubbles that are administered intravenously to the systemic circulation. Microbubbles have a high degree of echogenicity, which is the ability of an object to reflect the ultrasound waves. The echogenicity difference between the gas in the microbubbles and the soft tissue surroundings of the body is immense. Thus, ultrasonic imaging using microbubble contrast agents enhances the ultrasound backscatter, or reflection of the ultrasound waves, to produce a unique sonogram with increased contrast due to the high echogenicity difference. Contrast-enhanced ultrasound can be used to image blood perfusion in organs, measure blood flow rate in the heart and other organs, and has other applications as well.

Targeting ligands that bind to receptors characteristic of intravascular diseases can be conjugated to microbubbles, enabling the microbubble complex to accumulate selectively in areas of interest, such as diseased or abnormal tissues. This form of molecular imaging, known as targeted contrast-enhanced ultrasound, will only generate a strong ultrasound signal if targeted microbubbles bind in the area of interest. Targeted contrast-enhanced ultrasound can potentially have many applications in both medical diagnostics and medical therapeutics. However, the targeted technique has not yet been approved for clinical use; it is currently under preclinical research and development.

General features
There are a variety of microbubbles contrast agents. Microbubbles differ in their shell makeup, gas core makeup, and whether or not they are targeted.


 * Microbubble shell: selection of shell material determines how easily the microbubble is taken up by the immune system. A more hydrophilic material tends to be taken up more easily, which reduces the microbubble residence time in the circulation.  This reduces the time available for contrast imaging.  The shell material also affects microbubble mechanical elasticity.  The more elastic the material, the more acoustic energy it can withstand before bursting (McCulloch et al., 2000).  Currently, microbubble shells are composed of albumin, galactose, lipid, or polymers (Lindner, 2004).


 * Microbubble gas core: The gas core is the most important part of the ultrasound contrast microbubble because it determines the echogenicity. When gas bubbles are caught in an ultrasonic frequency field, they compress, oscillate, and reflect a characteristic echo- this generates the strong and unique sonogram in contrast-enhanced ultrasound.  Gas cores can be composed of air, or heavy gases like perfluorocarbon, or nitrogen (Lindner, 2004).  Heavy gases are less water-soluble so they are less likely to leak out from the microbubble to impair echogenicity (McCulloch et al., 2000).  Therefore, microbubbles with heavy gas cores are likely to last longer in circulation.

Optison, a Food and Drug Administration (FDA)-approved microbubble made by GE Healthcare, has an albumin shell and octafluoropropane gas core. The second FDA-approved microbubble, Levovist, made by Schering, has a lipid/galactose shell and an air core. (Lindner, 2004)

Regardless of the shell or gas core composition, microbubble size is fairly uniform. They lie within in a range of 1-4 micrometres in diameter. That makes them smaller than red blood cells, which allows them to flow easily through the circulation as well as the microcirculation.

Targeted microbubbles
Targeted microbubbles are under preclinical development. They retain the same general features as untargeted microbubbles, but they are outfitted with ligands that bind specific receptors expressed by cell types of interest, such as inflamed cells or cancer cells. Current microbubbles in development are composed of a lipid monolayer shell with a perflurocarbon gas core. The lipid shell is also covered with a polyethylene glycol (PEG) layer. PEG prevents microbubble aggregation and makes the microbubble more non-reactive. It temporarily “hides” the microbubble from the immune system uptake, increasing the amount of circulation time, and hence, imaging time (Klibanov, 2005). In addition to the PEG layer, the shell is modified with molecules that allow for the attachment of ligands that bind certain receptors. These ligands are attached to the microbubbles using carbodiimide, maleimide, or biotin-streptavidin coupling (Klibanov, 2005). Biotin-streptavidin is the most popular coupling strategy because biotin’s affinity for streptavidin is very strong and it is easy to label the ligands with biotin. Currently, these ligands are monoclonal antibodies produced from animal cell cultures that bind specifically to receptors and molecules expressed by the target cell type. Since the antibodies are not humanized, they will elicit an immune response when used in human therapy. Humanizing antibodies is an expensive and time-intensive process, so it would be ideal to find an alternative source of ligands, such as synthetically manufactured targeting peptides that perform the same function, but without the immune issues.

How contrast-enhanced ultrasound works
There are two forms of contrast-enhanced ultrasound, untargeted (used in the clinic today) and targeted (under preclinical development). The two methods slightly differ from each other.

Untargeted CEUS
Untargeted microbubbles, such as the aforementioned Optison or Levovist, are injected intravenously into the systemic circulation in a small bolus. The microbubbles will remain in the systemic circulation for a certain period of time. During that time, ultrasound waves are directed on the area of interest. When microbubbles in the blood flow past the imaging window, the microbubbles’ compressible gas cores oscillate in response to the high frequency sonic energy field, as described in the ultrasound article. The microbubbles reflect a unique echo that stands in stark contrast to the surrounding tissue due to the orders of magnitude mismatch between microbubble and tissue echogenicity. The ultrasound system converts the strong echogenicity into a contrast-enhanced image of the area of interest. In this way, the bloodstream’s echo is enhanced, thus allowing the clinician to distinguish blood from surrounding tissues.

Targeted CEUS
Targeted contrast-enhanced ultrasound works in a similar fashion, with a few alterations. Microbubbles targeted with ligands that bind certain molecular markers that are expressed by the area of imaging interest are still injected systemically in a small bolus. Microbubbles theoretically travel through the circulatory system, eventually finding their respective targets and binding specifically. Ultrasound waves can then be directed on the area of interest. If a sufficient number of microbubbles have bound in the area, their compressible gas cores oscillate in response to the high frequency sonic energy field, as described in the ultrasound article. The targeted microbubbles also reflect a unique echo that stands in stark contrast to the surrounding tissue due to the orders of magnitude mismatch between microbubble and tissue echogenicity. The ultrasound system converts the strong echogenicity into a contrast-enhanced image of the area of interest, revealing the location of the bound microbubbles (Klibanov, 1999). Detection of bound microbubbles may then show that the area of interest is expressing that particular molecular, which can be indicative of a certain disease state, or identify particular cells in the area of interest.

Applications of contrast-enhanced ultrasound
Untargeted contrast-enhanced ultrasound is currently applied in echocardiography. Targeted contrast-enhanced ultrasound is being developed for a variety of medical applications.

Untargeted CEUS
Untargeted microbubbles like Optison and Levovist are currently used in echocardiography.
 * Organ Edge Delineation: microbubbles can enhance the contrast at the interface between the tissue and blood. A clearer picture of this interface gives the clinician a better picture of the structure of an organ.  Tissue structure is crucial in echocardiograms, where a thinning, thickening, or irregularity in the heart wall indicates a serious heart condition that requires either monitoring or treatment.
 * Blood Volume and Perfusion: contrast-enhanced ultrasound holds the promise for (1) evaluating the degree of blood perfusion in an organ or area of interest and (2) evaluating the blood volume in an organ or area of interest. When used in conjunction with Doppler Ultrasound, microbubbles can measure myocardial flow rate to diagnose valve problems.  And the relative intensity of the microbubble echoes can also provide a quantitative estimate on blood volume.

Targeted CEUS

 * Inflammation: in inflammatory diseases such as Crohn’s disease, atherosclerosis, and even heart attacks, the inflamed blood vessels specifically express certain receptors like VCAM-1, ICAM-1, E-selectin. If microbubbles are targeted with ligands that bind these molecules, they can be used in contrast echocardiography to detect the onset of inflammation.  Early detection allows the design of better treatments.
 * Cancer: cancer cells also express a specific set of receptors, mainly receptors that encourage angiogenesis, or the growth of new blood vessels. If microbubbles are targeted with ligands that bind receptors like VEGF, they can non-invasively and specifically identify areas of cancers.
 * Gene Delivery: Vector DNA can be conjugated to the microbubbles. Microbubbles can be targeted with ligands that bind to receptors expressed by the cell type of interest.  When the targeted microbubble accumulates at the cell surface with its DNA payload, ultrasound can be used to burst the microbubble.  The force associated with the bursting may temporarily permeablize surrounding tissues and allow the DNA to more easily enter the cells.
 * Drug Delivery: drugs can be incorporated into the microbubble’s lipid shell. The microbubble’s large size relative to other drug delivery vehicles like liposomes may allow a greater amount of drug to be delivered per vehicle.  By targeted the drug-loaded microbubble with ligands that bind to a specific cell type, microbubble will not only deliver the drug specifically, but can also provide verification that the drug is delivered if the area is imaged using ultrasound.

Recent microbubble targeting history
Microbubbles can be used in various contrast-enhanced ultrasound applications, as shown above. The area of greatest area of promise and growth lies in targeted contrast-enhanced ultrasound. Current microbubble targeting strategies produce low adhesion efficiencies at high vessel shear stresses of physiological relevance. This means that only a small fraction of microbubbles injected into the test subject actually binds to the molecular markers of interest (Takalkar et al., 2004). This is one of the main issues preventing targeted contrast-enhanced ultrasound’s jump from bench to bedside.

There has been an increasing interest in the biomedical research community to enhance the adhesion efficiency of microbubble contrast agents in order to realize targeted contrast-enhanced ultrasound’s immense diagnostic and therapeutic potentials. Scientists have outfitted microbubbles with monoclonal antibodies that bind endothelial markers of inflammation, specifically the cell adhesion molecules P-selectin, ICAM-1, and VCAM-1. They showed that these complexes enable targeted ultrasound imaging of inflammation (Lindner, 2004). But, the aforementioned efficiency of microbubble adhesion to the molecular target was poor and a large fraction of microbubbles that bound to the target rapidly detached, especially at high shear stresses of physiological relevance (Takalkar et al., 2004). Effective contrast-enhanced ultrasound requires efficient microbubble binding at the area of imaging interest (Klibanov, 1999).

Leukocytes possess high adhesion efficiencies, partly due to a dual-ligand selectin-integrin cell arrest system (Eniola et al., 2003). One ligand:receptor pair (PSGL-1:selectin) has a fast bond on-rate to slow the leukocyte and allows the second pair (integrin:immunoglobulin superfamily), which has a slower on-rate but slow off-rate to arrest the leukocyte, kinetically enhancing adhesion.

Several research groups have taken advantage of this concept. Eniola and Hammer at the University of Pennsylvania applied dual-ligand targeting of distinct receptors to polymer microspheres for drug delivery and reported an increase in microsphere binding (Eniola and Hammer, 2005). Similarly, Weller and colleagues at the University of Pittsburgh used microbubbles targeted to bind two distinct receptors and showed increased microbubble adhesion strength (Weller et al., 2005). Biomimcry of the leukocyte’s selectin-integrin cell arrest system has also been investigated in the context of improving microbubble adhesion efficiency at the University of Virginia (Rychak et al., 2005). All three research groups showed that dual-targeted microbubbles showed enhanced adhesion compared to single-targeted microbubbles. Though this strategy markedly improves upon prior adhesion, it is still less than ideal. The adhesion efficiency must be higher to allow clinical use of targeted contrast-enhanced ultrasound.

Advantages of contrast-enhanced ultrasound
On top of the strengths mentioned in the medical sonography entry, contrast-enhanced ultrasound adds these additional advantages:
 * The body is 90% water, and therefore, acoustically homogeneous. Blood and surrounding tissues have similar echogenicities, so it is also difficult to clearly discern the degree of blood flow, perfusion, or the interface between the tissue and blood using traditional ultrasound (Lindner, 2004).
 * Ultrasound imaging allows real-time evaluation of blood flow (Lindner et al., 2002).
 * Ultrasonic molecular imaging is safer than molecular imaging modalities such as radionuclide imaging because it does not involve radiation (Lindner et al., 2002).
 * Alternative molecular imaging modalities, such as MRI, PET, and SPECT are very costly. Ultrasound, on the other hand, is very cost-efficient and widely available (Klibanov, 1999).
 * Since microbubbles can generate such strong signals, a lower intravenous dosage is needed, micrograms of microbubbles are needed compared to milligrams for other molecular imaging modalities such as MRI contrast agents (Klibanov, 1999).
 * Targeting strategies for microbubbles are versatile and modular. Targeting a new area only entails conjugating a new ligand.

Disadvantages of contrast-enhanced ultrasound
In addition to the weaknesses mentioned in the medical sonography entry, contrast-enhanced ultrasound suffers from the following disadvantages:
 * Microbubbles don’t last very long in circulation. They have low circulation residence times because they either get taken up by immune system cells or get taken up by the liver or spleen even when they are coated with PEG (Klibanov, 1999).
 * Ultrasound produces more heat as the frequency increases, so the ultrasonic frequency must be carefully monitored.
 * Microbubbles burst at low ultrasound frequencies and at high mechanical indices (MI), which is the measure of the acoustic power output of the ultrasound imaging system. Increasing MI increases image quality, but there are tradeoffs with microbubble destruction.  Microbubble destruction could cause local microvasculature ruptures and hemolysis (Klibanov, 2005).
 * Targeting ligands can be immunogenic, since current targeting ligands used in preclinical experiments are derived from animal culture (Klibanov, 2005).
 * Low targeted microbubble adhesion efficiency, which means a small fraction of injected microbubbles bind to the area of interest (Takalkar et al., 2004). This is one of the main reasons that targeted contrast-enhanced ultrasound remains in the preclinical development stages.