Microbubble Contrast Agents Using Ultrasound


Deepika Maliwal 1* and Vidyasagar Patidar 2

1Dept. of Pharma. Chemistry, 2Dept. of Pharmaceutics, B. R. Nahata College of Pharmacy, Mandsaur




Contrast-enhanced ultrasound (CEUS) is the application of ultrasound contrast agents to traditional medical sonography

Ultrasound contrast agents are gas-filled micro-bubbles that are administered intravenously to the systemic circulation. The field of Contrast-enhanced ultrasound is expanding rapidly with exciting new applications. While ultrasound contrast agents were initially used to overcome insufficient transcranial bone windows for identification of the basal cerebral arteries, new-generation micro-bubbles in combination with very sensitive contrast-specific ultrasound techniques now enable real-time visualization of stroke. This article will provide a review of recent and emerging developments in ultrasound technology and contrast-specific imaging techniques.


KEY WORDS Contrast-enhanced ultrasound, ultrasound contrast agents.



Contrast-enhanced ultrasound (CEUS) is the

application of ultrasound contrast agents to traditional medical sonography. Ultrasound contrast agents are gas-filled micro-bubbles that are administered intravenously to the systemic circulation. Micro- bubbles 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 micro-bubbles and the soft tissue surroundings of the body is immense. Thus, ultrasonic imaging using micro-bubble 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. Targeting ligand that bind to receptors characteristic of intravascular diseases can be conjugated to micro-bubbles, enabling the micro- bubble 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 microb-ubbles 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


Microbubble Contrast Agents:

General features

There are a variety of micro-bubbles contrast agents. Micro-

bubbles differ in their shell makeup, gas core makeup, and whether or not they are targeted.

Micro-bubble shell: selection of shell material determines how easily the micro-bubble is taken up by the immune system. A more hydrophilic material tends to be taken up more easily, which reduces the micro- bubble residence time in the circulation. This reduces the time available for contrast imaging. The shell material also affects micro-bubble mechanical elasticity. The more elastic the material, the more acoustic energy

it can withstand before bursting1. Currently, micro- bubble shells are composed of albumin, galactose, lipid,

or polymers 2.


Micro-bubble gas core: The gas core is the most important part of the ultrasound contrast micro-bubble 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 nitrogen2. Heavy gases are less water-soluble so they are less likely to leak out from the micro-bubble to impair echogenicity1 .Therefore, micro-bubbles with heavy gas cores are likely to last longer in circulation.


Safety of micro-bubbles

As with all pharmaceutical agents, micro-bubble agents have

to pass rigorous safety tests in order for them to be licensed. Extensive trials have established an excellent overall safety record with few significant adverse effects. However, there are some concerns that disruption of their outer shells produces local bioeffects such as sonoporation (subcellular membrane damage) and cell lysis at diagnostic frequencies and that perfluorocarbon gases enhance these effects. Despite this, no effects of these processes have been observed in humans. Concern about administering volumes of gas into the blood stream has been shown not to be an issue as the amount given is under 200 mL, too small to exhibit an effect. Overall, it has been shown that the safety of micro- bubbles compares favourably to that of conventional radiographic contrast agents and those used in contrast- enhanced MRI 3.


Working Of Contrast-Enhanced Ultrasound:

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 bloodstreams 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 ligand 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 4. 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

Crohns 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 microbubbles lipid shell. The microbubbles 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.


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 2

Ultrasound imaging allows real-time evaluation of blood flow 5.

Ultrasonic molecular imaging is safer than molecular imaging modalities such as radionuclide imaging because it does not involve radiation5.

Alternative molecular imaging modalities, such as

MRI, PET, and SPECT are very costly. Ultrasound, on the other hand, is very cost- efficient and widely available4.

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 4.

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 dont 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 4.

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 6

Targeting ligands can be immunogenic, since current targeting ligands used in preclinical experiments are derived from animal culture 6 .

Low targeted microbubble adhesion efficiency, which means a small fraction of injected microbubbles bind to the area of interest 7 This is one of the main reasons that targeted contrast-enhanced ultrasound remains in the preclinical development stages.



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Received on 26.06.2008 Modified on 10.07.2008

Accepted on 10.08.2008 RJPT All right reserved

Research J. Pharm. and Tech. 1(3): July-Sept. 2008; Page 152-154