This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liang, H-D Articles by Blomley, M J K Search for Related Content PubMed PubMed Citation Articles by Liang, H-D Articles by Blomley, M J K

The role of ultrasound in molecular imaging

Ultrasound Group, Imaging Sciences Department, Imperial College London, Clinical Sciences Centre, Hammersmith Campus, Du Cane Road, London W12 0HS, UK

Correspondence: Dr M J K Blomley

	Abstract

Top Abstract Introduction Applications to molecular... Applications to molecular... Applications to molecular... Applications to molecular... Summary References

 
Ultrasound has received less attention than other imaging modalities for molecular imaging, but has a number of potential advantages. It is cheap, widely available and portable. Using Doppler methods, flow information can be obtained easily and non-invasively. It is arguably the most physiological modality, able to image structure and function with less sedation than other modalities. This means that function is minimally disturbed, and multiple repeat studies or the effect of interventions can easily be assessed. High frame rates of over 200 frames a second are achievable on current commercial systems, allowing for convenient cardiac studies in small animals. It can be used to guide interventional or invasive studies, such as needle placement. Ultrasound is also unique in being both an imaging and therapeutic tool and its value in gene therapy has received much recent interest. Ultrasound biomicroscopy has been used for in utero imaging and can guide injection of virus and cells. Ultrahigh frequency ultrasound can be used to determine cell mechanical properties. The development of microbubble contrast agents has opened many new opportunities, including new functional imaging methods, the ability to image capillary flow and the possibility of molecular targeting using labelled microbubbles.

	Introduction

Top Abstract Introduction Applications to molecular... Applications to molecular... Applications to molecular... Applications to molecular... Summary References

 
Ultrasound is the most widely used cross-sectional imaging modality worldwide [1]. Important recent developments include microbubble contrast agents, quantitative approaches and new signal processing and display methods. In this review, we describe some recent development of the technology of ultrasound relevant to molecular imaging. These include a number of promising ways of imaging the microcirculation using ultrasound, advances in high and ultrahigh frequency ultrasound, and an overview of the potential of microbubble contrast agents and ultrasound-mediated gene transfection. This review article will first describe some basic principles of ultrasound and then illustrate some applications relevant to molecular imaging. Key areas to be discussed will be developments in high frequency ultrasound, angiogenesis imaging, targeted microbubbles and microbubble ultrasound mediated gene and drug delivery.

Ultrasound refers to the sound at a frequency higher than 20 kHz, and may be broken down by the frequencies used. Clinical diagnostic ultrasound scanners use frequencies in the range of 1 MHz to 20 MHz. Ultrasound biomicroscopy (UBM) typically operates at frequencies of 40 MHz to 200 MHz, while scanning acoustic microscopy (SAM) uses frequencies higher than 200 MHz. Currently almost all commercial systems operate in the clinical diagnostic range, although commercial biomicroscopy is now a reality (http://www.visualsonics.com). It is crucial to understand that resolution improves with frequency, while penetration dramatically decreases with frequency. Therefore, the choice of ultrasound frequency is a balance between resolution and penetration depth, with higher frequencies used for more superficial structures.

Pulses of ultrasound are sent out in trains from arrays of transducers (probes) and the returning echoes analysed to build up a two-dimensional image of the plane being scanned and displayed using a grey-scale display to code the intensity of the returning echoes. This produces the B-mode (brightness modulated) images. If sound is reflected from a moving structure such as a red blood cell, it will Doppler shift in frequency according to the velocity between the structure and the probe. The frequency shift can be displayed as a spectral trace or colour image representing the movement of erythrocytes.

In colour Doppler imaging, frequency shifts from different areas of the image are analysed and displayed as a colour overlay. Typically, positive Doppler shift signals corresponding to flow towards the transducer are displayed at the red end of the spectrum and flow away from the transducer at the blue end. The amount of vascularity in a lesion and the arrangement of blood vessels may help distinguish some tumours from benign masses. Power Doppler imaging shows the intensity of the Doppler signal displayed as a colour overlay on the grey-scale image. This is much less dependent on flow direction and generally more sensitive to low flow signals but gives no information on flow direction or velocity.

Ultrasound contrast agents (UCAs) can be used to improve imaging by introducing a material with different acoustic properties from that of tissues [2]. The most common approach is the use of intravenous injections of small air or gas bubbles (microbubbles) that boost the Doppler signal from blood vessels. The original impetus for their development (to enhance Doppler signals) has been largely superseded by a number of niche "microbubble-specific" applications.

Microbubbles work not only because they provide a strongly reflective blood/gas interface, but also because they resonate in the ultrasound beam, rapidly contracting and expanding in response to the pressure changes of a sound wave. This makes them several orders of magnitude more reflective than normal blood. In this way, they enhance both normal grey-scale and flow-mediated Doppler signals. At higher acoustic powers, although still well within the recommended limits for diagnostic scanning, ultrasound destroys many microbubbles relatively easily. Some microbubbles are tissue-specific (for example, some show tropism for the reticuloendothelial system) and this can also be exploited in a number of emerging applications. For example, many malignant liver lesions stand out as defects when liver specific microbubbles are imaged, and their visibility can be greatly increased [3].

Microbubbles can help in visualizing flow in smaller vessels even to the capillary level, especially when harmonic and other non-linear imaging methods are used. As with conventional Doppler signals, the changes produced by microbubbles can also be quantified for functional imaging purposes.

Microbubbles can be quantified in passive and active imaging approaches. In passive approaches, low acoustic power is used to observe the enhancement effects without disrupting microbubbles. The intensity changes produced by them can be quantified and in some situations are proportional to relative microbubble concentration [4]. The active approach uses a transient pulse of high acoustic power to destroy microbubbles and then observe refilling into an area of interest during an infusion. One method is to repeatedly pulse ultrasound at varying triggering intervals, waiting for a steady state, and study the relationship between steady-state enhancement levels and triggering delay settings [5, 6]. Another is to momentarily scan with high acoustic power and then scan using very low power settings, watching the microbubble refilling rates [7]. These re-perfusion or kinetic methods have the advantage of measuring novel indices that cannot be measured in any other way, such as microcirculatory flow speed. In principle, both methods can give information on microcirculatory flow speeds and fractional vascular volume of a tissue. Quantitative flow data can be extracted from regions within the image plane and these results displayed as colour overlays on the original image data.

Ultrasound is unique in being both an imaging and therapeutic tool. Ultrasound can cause tissue bioeffects, including tissue heating and shock waves produced by the oscillatory behaviour or collapse of minute bubbles containing gas or vapour (i.e. cavitation). At the energies permitted for normal diagnostic scanning, bioeffects are not thought to be a clinical problem. At higher powers, however, bioeffects ranging from minor tissue heating to tissue ablation (focused ultrasound surgery) can be seen. The delivery of molecules and genes into cells can be enhanced, mainly through the production of transient non-lethal perforations in cell membranes. These effects are increased when gas bodies, such as microbubbles are present, and the use of microbubble ultrasound for drug delivery and gene therapy is receiving great interest currently. In addition, a number of microbubbles have been engineered, which show specificity for various molecular targets by attaching ligands such as antibodies or peptides, although at present these are still at the early pre-clinical stage. These rapidly developing subjects are discussed later in this article.

	Applications to molecular imaging: developments in high frequency ultrasound

Top Abstract Introduction Applications to molecular... Applications to molecular... Applications to molecular... Applications to molecular... Summary References

 
There have been dramatic advances in high frequency ultrasound for animal and tissue imaging in recent years. This large subject will be reviewed by discussing first the use of commercial clinical systems, then biomicroscopy and finally ultrahigh frequency scanning acoustic microscopy.

Pre-clinical ultrasound applications with commercial clinical systems
Ultrasound is widely used for animal imaging, as it is cheap, portable, real-time and allows for convenient intervention and biopsy. One of the most promising newer uses is for echocardiography in small animals where high temporal resolution is crucial (a mouse heart rate beats typically 450–600 times a minute). The physiological nature of ultrasound lends itself well to this type of application as only minimal sedation is needed, and some investigators have even been able to train animals to tolerate ultrasound without any sedation. Using various methods (the use of a small superficial "zoom" box in the near field is especially helpful) frame rates of over 200 frames per second can be achieved on commercial systems used for patients without modification.

Ultrasonic biomicroscopy (40–200 MHz)
Turnbull et al demonstrated the first use of UBM for non-invasive in utero imaging of live mouse embryos and further showed the method to be effective in detecting and quantifying the midhindbrain deletion associated with a null mutation of Wnt-1 [8]. More recent investigations in this area have focused on high frequency ultrasound imaging and Doppler methods for examing mouse cardiovascular development [9, 10].

A number of genes have been identified in the mouse that are critical for normal development of the cardiovascular system. The use of UBM for cardiovascular assessment of cardiac structure and function in mouse embryos has been reported by Srinivasan et al [10].

Figure 1 shows the complete ventricular septation and visible mitral value leaflets of the heart at day 13.5.

View larger version (151K): [in this window] [in a new window]   Figure 1. A four-chamber view of the heart at day 13.5 showing complete ventricular septation and visible mitral valve leaflets. Note that at these high frequencies the blood pool appears relatively echogenic. Reprinted from [11] with permission.

View larger version (79K): [in this window] [in a new window]   Figure 2. 40 MHz ultrasound biomicroscopy (UBM)-guided continuous wave (CW) Doppler measurements of umbilical blood flow. (a) In utero UBM image of an E12.5 mouse embryo (E), showing the Doppler sample volume (white box) positioned over the umbilical artery (A) and vein (V). The placenta is labelled as P. The scale bar is 1 mm. (b) CW Doppler waveform demonstrating arterial (A) flow towards the transducer (positive) and venous flow (V) away from the transducer (negative). Reprinted from [9] with permission.

The availability of real-time, in utero imaging of mouse embryos has made it possible to perform UBM-guided injections of cells, viruses or other agents into precise locations in the developing embryo over a range of pre-determined stages [13–16].

High frequency (Doppler) ultrasound offers the potential to assess angiogenesis non-invasively and to monitor the effects of antivascular therapy on tumour blood flow [17]. Continuous wave (CW), pulsed and colour flow Doppler in the 50 MHz range are now being evaluated. Detection of microvasculature to about 10 µm size vessls is possible [18]. The subject of assessing neovascularity using ultrasound is discussed in more detail later.

UBM has also been used to characterize cells, suggesting this imaging modality could differentiate between different types of cell morphology [19, 20]. Apoptosis (programmed cell death) plays a significant role in both normal and disease-related biological processes. Biomicroscopy data show more than doubled ultrasound backscatter signal from apoptotic cells in comparison with viable cells, whereas heat-killed cells exhibit an intermediate level of ultrasound backscatter [21, 22]. The different image intensities could arise from the differences in ultrasound scattering due to highly condensed cluster of nuclear material in the apoptotic cells [23].

Scanning acoustic microscopy (200 MHz and higher)
Amongst methods for determining the mechanical properties of living cells, SAM provides some intriguing potential, although it has not yet reached maturity as a pathological tool. Relative changes in cytoplasmic forces may be resolved with a time resolution of about 5 s, and exact determination of mechanical properties requires a series of five or six images [24, 25]. It provides high spatial resolution in the range of 3 µm. Its minimal invasiveness results from the very high frequency of ultrasound used, which does not generally cause damage or disturbance to the cells [26].

The indices measured with SAM include the speed of sound in tissue, attenuation, impedance and cellular dimensions (Figure 3). Methods used are based on shear wave propagation [27] or on measurements of the effects on reflected sound produced by varying focus positions [28] or frequency [29]. The computation of cell properties from acoustic microscope-generated signals requires an estimation of the cell thickness profile [30–35]. The cell profile is traditionally computed by counting the interference rings and estimating the value of the longitudinal wave speed in the cell. Kundu et al used this information to arrive at a rough estimate of the probable upper and lower bounds of the cell thickness and then adopted a simplex inversion algorithm to predict the cell thickness accurately, along with other cell parameters [34, 35].

View larger version (53K): [in this window] [in a new window]   Figure 3. (a) An ultrasonic image generated by plotting the variation of the intensity of the reflected beam. The cells are XTH-2 cells, originating from Xenopus tadpole heart endothelium. The straight line on the image is the scan line along which cell properties are computed. (b) Predicted wave speed (continuous line) and attenuation (dotted line) variation along the scan line. Reprinted from [29] with permission.

SAM can in principle be applied for intraoperative pathological examination, as SAM does not require any special staining technique. The spatial resolution of SAM at 600 MHz is about 3 µm, comparable with optical methods [36, 37]. SAM at 200 MHz has been used to classify tumour types in stomach [38] and kidney [28].

	Applications to molecular imaging: imaging angiogenesis

Top Abstract Introduction Applications to molecular... Applications to molecular... Applications to molecular... Applications to molecular... Summary References

 
Conventional ultrasound can be used to image the microcirculation using both Doppler and microbubble methods. Power Doppler can be quantified to give an estimate of relative fractional vascular volume and a number of studies have shown indices derived in this way may be clinically useful [39]. Various manufacturer and offline measurement packages have been described or are available, and the interested reader is referred elsewhere for a fuller description [5]. Microbubbles, by raising the signal from smaller vessels, can show flow down to the level of the microcirculation.

Power Doppler methods
Increased understanding of the role of angiogenesis in malignancy and its treatment has heightened interest in understanding the relationship between the Doppler signals and the microcirculation. Several researchers have studied the relationship between histological indices of angiogenesis (notably microvessel density count (MVD)) and indices derived from Doppler ultrasound. Power Doppler may be seen as a machine processed map of moving reflectors. It should therefore be possible to quantify it and measure relative blood volume. In general, it would appear that tumours showing increasing MVD show higher Doppler-derived indices, but current Doppler methods often perform relatively poorly when directly correlated with measures of tumour angiogenesis indices such as MVD [40]. A more promising clinical application at the moment is imaging the response of a tumour blood supply to cancer therapy using estimates based on the fractional vascular volume [41]. The relative simplicity and ease of use of ultrasound would potentially make this a very appealing method of following cancer therapy in patients.

Another application is the prediction of metastastic spread and survival, especially in colorectal cancer. One approach is to assess the Doppler vascularity of the primary tumour, on the hypothesis that the more vascular tumours are, the more they are likely to metastasize. An alternative approach to detecting early metastatic disease is to try and detect vascular changes in the liver blood supply, which may indicate a propensity to develop visible metastases at a later date.

Microbubble methods
Most of the earlier research in this area has been based on studying enhancement in larger vessels, for example, by scanning in power Doppler mode after an injection of microbubbles. Some studies showed encouraging evidence that longer enhancement times were seen in malignant as opposed to benign breast lesions [42]. It is very likely, however, that the use of newer "active" approaches, such as the "re-perfusion kinetic" method will offer considerable advantages, as they allow capillary imaging and thereby assessing tumour neovascularity directly. An encouraging study by Halpern and colleagues using transrectal scanning of the prostate showed that the visibility of cancers could be greatly improved by the abnormal enhancement seen using a re-perfusion kinetic method [43].

One area, however, where functional methods are moving beyond the research arena and becoming established clinical tools is in the liver. Haemangiomas, which are common incidental "nuisance" findings on ultrasound, show a characteristic pattern of peripheral globular enhancement with progressive centripetal infilling on angiography, CT and MRI. Similar findings can be seen using microbubble ultrasound, particularly if care is taken to avoid microbubble disruption by using either intermittent/low power scanning or newer more robust microbubbles [44, 45]. The use of microbubble enhanced ultrasound should produce cost benefits and be more convenient for the patient as well as reduce the ionizing radiation burden. A new microbubble mode (Microvascular Imaging: MVI, Philips Ultrasound, Bothwell, WA) integrates the microbubble signal within a tumour over time, providing a map of tumour vessels throughout a lesion (Figure 4).

View larger version (148K): [in this window] [in a new window]   Figure 4. This is the processed image demonstrating the microvascular map (based on integrating microbubble signals) within a 2.8 cm breast lesion (in a patient diagnosed with grade 2 invasive ductal carcinoma). Note the hypoechoic central region, which is assumed to represent a hypovascular area within the lesion). Image supplied by Anand Rattansingh, Research Radiographer, Imperial College and Hammersmith Hospital, with permission.

Another strategy for imaging angiogenesis is the use of targeted microbubbles, and this is the subject of the next section.

	Applications to molecular imaging: targeted ultrasound contrast agents

Top Abstract Introduction Applications to molecular... Applications to molecular... Applications to molecular... Applications to molecular... Summary References

 
Several strategies have been developed to promote targeting of microbubble contrast agents to specific regions of disease. This could potentially be useful both for imaging or therapy, as described in the next section of this review. The first approach takes advantage of inherent chemical or electrostatic properties of the microbubble shell, resulting in arrest of microbubbles within the microcirculation. This method relies on the disease related upregulation of receptors that bind non-specifically to either albumin or lipid components of the microbubble shell [50]. The second method is also referred to "active targeting" or "specific targeting" [2]. It uses the deliberate attachment of specific antibodies or other ligands to the microbubble surface, leading to the accumulation of targeted contrast agents at a specific site due to the use of adhesion ligands, which recognise disease antigens. A variety of adhesive ligands are being explored to specifically bind to cellular receptors indicative of disease. Potential ligands include antibodies, peptides, polysaccharides [51]. Most targeted ultrasound contrast agents are microbubbles, but other vehicles can be used including acoustically active liposomes and perfluorocarbon emulsions, and these will be considered in turn.

Microbubbles that target the reticuloendothelial system
Some microbubbles are tropic to the reticuloendothelial system, although the properties that make some agents do this are still not known. It is known that in some cases this is through direct phagocytosis by the Kupffer cells in the liver and spleen. The phospholipid shell contrast agent AF0150 (Imavist; Alliance Pharmaceutical, San Diego, CA) is one such agent [52]. In vivo colour Doppler imaging at 5–10 MHz illustrated the destruction of microbubbles captured in macrophages in the spleen and liver. Hepatomas were detected as an anechoic region during this acoustic interrogation. The sensitivity of detection of VX-2 liver tumours in rabbit models increased from 33% to 100% with late stage imaging of the contrast agent. Similarly, the sensitivity of detection of hepatocellular carcinomas was increased from 20% to 70% in five woodchucks with the use of the agent [53].

Some commercial agents also have liver specific properties such as the agent Levovist (Schering, Berlin, Germany), although it is not clear if this is through phagocytosis or just sinusoidal adherence in liver passage. This can be used not only to improve the detection of liver malignancies, but also to characterize them. If a liver lesion is scanned during the liver phase of Levovist, marked differences can be seen between malignant lesions, which usually appear as a defect and most benign lesions (focal fat or fatty sparing, focal nodular hyperplasia and many haemangiomas), which show uptake, presumably because they either contain Kupffer cells or show delayed vascular retention in the case of haemangiomas [3].

Microbubbles that target inflammation
During inflammation, a cascade of molecular signals results in leukocyte migration from the blood pool to the extravascular space. Adhesion molecules such as P-selectin are rapidly expressed on the surface of activated endothelial cells, and leukocytes are tethered to the endothelial surface of small venules through selectin-mediated adhesion. P-selectin mediates leukocyte attachment through interaction with carbohydrate ligands on the cellular surface. Leukocytes roll along the venular surface and eventually become firmly adherent. Adhesion during rolling is mediated by selectin molecules, including the P- and E-selectins expressed on endothelial cells, L-selectins on leukocytes, and other intercellular adhesion molecules such as ICAM-1 and VCAM-1.

Albumin shelled UCA are strongly adherent to activated endothelial cells through the first type of non-specific binding referred to above [54]. Optison and MP1950 (Amersham, Oslo, Norway) adhere to leukocytes activated by inflammatory response [50, 55, 56]. Albumin bubble–leukocyte adhesion is mediated by the ₂ integrin Mac-1, which typically plays a role in the adhesion of leukocytes to proteins found in the extracellular matrix and serum [57, 58].

Lindner et al demonstrated the accumulation of microbubbles with adherent leukocytes in venules in the inflamed mouse cremaster muscle. Inflammation was artificially stimulated using either ischaemia–re-perfusion or tumour necrosis factor- (TNF-) [50]. Ultrasound images of the TNF- stimulated cremaster muscle after microbubble infusion demonstrate intense contrast enhancement from phagocytosed and adherent microbubbles retained at the site of inflammation. Dayton et al have shown that the damping effect of leukocyte cytoplasm on phagocytosed microbubbles results in an acoustic echo for a phagocytosed microbubble that is different than that from a free microbubble of the same size [59]. This result allows for selective detection of phagocytosed microbubbles.

Endothelial cells activated during the inflammatory response were targeted using a lipid shelled microbubble engineered with a monoclonal antibody to ICAM-1 [60]. This ligand, expressed on the surface of activated endothelial cells, is an early indicator of atherosclerosis [51]. A 40-fold increase in the spatial extent of microbubble adhesion occurred with targeted microbubbles, compared with a non-targeted control under static conditions [60]. A second approach to active targeting of inflammation involves antibodies to P-selectin conjugated to a phospholipid shelled microbubble [61]. Using P-selectin targeting, a significant increase in accumulation of microbubbles in the injured mouse kidney was measured after a cycle of ischaemia followed by re-perfusion [61].

Microbubbles that target tumours
Passive targeting of C6 glioma and L9 glioscarcoma brain tumours by phagocytosis of microbubbles has been observed by Barbarese et al using fluorescently-labelled lipid-coated microbubbles [62]. The microbubble concentration is increased over time after rat tail vein injection, with the fluorescence intensity of tumour cells increasing almost 300% over 30 min, compared with control rats, which were administered the fluorescent label without microbubbles. Passive targeting can also be used to deliver therapeutic drugs to brain tumours and brain injury sites [63, 64].

Molecular markers of angiogenesis can also be targeted. One recent study used a lipid-based microbubble contrast agent conjugated of echistatin to the shell surface targeted to _v3. This integrin is highly expressed by angiogenic endothelium. Contrast enhanced ultrasound signal from the targeted microbubbles was greatest at the periphery of tumours [65].

Microbubbles that target the lymphatic system
Some microbubbles, such as the submicron agents M1134 and M1136 (Point Biomedical, San Carlos, CA) and also some larger microbubbles can enter the lymphatic system after injection into the interstitium. The agents accumulate at the sentinel lymph node and typically do not pass to successive nodes. They undergo significant expansion during acoustic interrogation, resulting in a strong acoustic echo [66].

Microbubbles that target thrombus
Unger et al [67], Wright et al [68], Takeuchi et al [69] and Schumann et al [70] demonstrate the efficacy of lipid-shelled microbubble specifically designed to target thrombus. These microbubbles, MRX-408 or MRX-408A1 (ImaRx Inc., Tucson, AZ) incorporate the Arg-Gly-Asp (RGD) peptide sequence in the microbubble shell. The RGD sequence is adherent to the receptors for fibrinogen and von Willebrand factor, as well as several other integrins and related heterodimeric proteins [71]. MRX-408 is targeted to the GPIIb/IIIa binding domain on activated platelets. In vitro 7.5 MHz ultrasound studies of artificially generated human thrombus demonstrated that MRX-408 enhanced the detectable extent of the clot an average of approximately nine fold, compared with a control non-targeted agent, and increased the probability of clot detection [67, 68]. Additionally, the use of MRX-408 in vivo enhanced detection of thrombus in the left atrial appendage in a canine model [69]. MRX-408 can also be used to enhance sonothrombolysis [72].

Targeted liposomal agents
Alkan-Onyuksel et al [73] and Demos et al [74–76] have demonstrated the application of echogenic liposomes, less than 1 µm in diameter, as a targeted contrast agent. Due to the small diameter, liposomal agents are not entrapped in the microvasculature of the lung, and therefore have a long circulating time. Additionally, the liquid-like composition of liposomes makes them more resistant to pressure and mechanical stress than encapsulated gas microbubbles. Another advantage of liposomes is that they can readily be conjugated to antibodies or other adhesion ligands, and thus are readily configured as targeted agents.

Liposomes can be made echogenic through lyophilization [73–77]. Lyophilization is believed to disrupt the lipid bilayers, which upon rehydration entrap small amounts of air. Entrapped air presents a significant acoustic impedance difference from tissue or plasma resulting echogenic liposomal agent. Alkan-Onyuksel et al have demonstrated that the ratio of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and cholesterol in the liposome can be optimized to produce agents that are echogenic both in vitro and in vivo [73].

Demos et al [74, 75] have demonstrated effective targeting of acoustically reflective liposomes to fibrin using anti-fibrinogen and anti-ICAM-1. The echogenicity of targeted microbubbles was examined in vitro using fibrin-coated paper or an excised artery from a Yucatan miniswine atherosclerosis model. Adhesion of targeted liposomes produced an increase in video intensity when observed using a 20 MHz intravascular ultrasound catheter. Further work by Demos et al demonstrated the effectiveness of targeted liposomes in vivo on miniswine atherosclerosis [76]. The animals were imaged using a 20 MHz intravascular ultrasound catheter or a linear 7.5 MHz conventional probe. The introduction of the targeted liposomal agent was shown to increase the intravascular video contrast of the target by 300% over blood and approximately 150% over agitated saline.

Targeted perfluorocarbon emulsion nanoparticles
Perfluorocarbon emulsion nanoparticles can be used as an ultrasound contrast agent. Due to their size and composition, these agents have the benefits of traditional gas encapsulated microbubble contrast agent and the liposomes [51, 78–82]. These nanoparticles are approximately a tenth of the size of many other UCAs (approximately 250 nm in diameter), and therefore avoid pulmonary entrapment. They consist of a lipid-encapsulated perfluorocarbon, which is a liquid at room temperature. The liquid composition makes it resistant to pressure and mechanical stress. However, due to their small size, their echogenicity is weak until they are deposited in a layer. This can be an advantage, as the low free-floating echogenicity results in a decreased background noise level. Hall et al [83–85] and Marsh et al [86] have developed theoretical models for estimating acoustic reflectivity of different perfluorocarbon nanoparticle formulations.

Application of these nanoparticle UCAs involved a three-step avidin–biotin linkage strategy [78]. In this strategy, a biotintylated target ligand, such as an antibody, protein, or other bioconjugate, was first administered and allowed to accumulate at the target site. This administration was followed by the biotin binding sites. Subsequent injection of the contrast agent, which itself has a biotin linker resulted in the adhesion of the contrast agent at the target site. Lanza et al have demonstrated the effectiveness of targeting clots both in vitro and in a canine carotid model with this method [78]. Preliminary in vitro experiments consisted of targeting porcine plasma clots, followed by ultrasound imaging with a 7.5 MHz linear transducer. With administration of the targeted agent, the average pixel video intensity increased approximately 12-fold. The evolution of clot enhancement due to agent deposition where the integrated echo intensity from a human clot increases approximately 8–12 dB over 20–60 min of exposure time.

Antibodies have been directly conjugated to the nanoparticle contrast agent before infusion [81, 87]. Nanoparticles with directly conjugated tissue factor antibody demonstrated acoustic enhancement similar to that demonstrated with the avidin–biotin targeting approach, with the advantage of one step targeting method. Flacke et al also demonstrate that these nanoparticles are effective as MR contrast agents [87]. The nanoparticles were formulated with the incorporation of gadolinium into the lipid shell for MRI. The ability to use either modality, or both simultaneously, provides a unique multimodality capability.

	Applications to molecular imaging: ultrasound-mediated gene therapy and drug delivery

Top Abstract Introduction Applications to molecular... Applications to molecular... Applications to molecular... Applications to molecular... Summary References

 
It has been known since the 1980s that ultrasound can enhance gene transfer by increasing cell permeability (sonoporation) [88]. Ultrasound has been demonstrated to deliver fluorescent dextran molecules [89–92], genetic material [93] and chemotherapeutic compounds [94] into viable cells. Therapeutic genes can be carried by microbubbles to the desired site and released by high acoustic power. Ultrasound energy alone can increase cell membrane permeability and thereby facilitate gene transfection. Ultrasound-mediated destruction of microbubbles can enhance this effect further. The destruction of microbubbles may facilitate transvascular delivery or intracellular deposition of therapeutic agents complexing the genes onto or within the microbubble can prevent them from inactivation or removal from the circulation. In principle this approach offers the prospect of safe site-specific delivery. Targeted microbubbles can improve efficiency even further.

The mechanism of sonoporation seems to be acoustic cavitation related [95, 96]. Acoustic cavitation involves the creation and oscillation of gas bubbles in a liquid. Normally, acoustic cavitation bubbles are believed to be generated from cavitation nucleii present in the tissues. At low ultrasound pressure field, these bubbles then shrink and grow in size due to rectified diffusion during ultrasound exposure, oscillating in response to the subsequent high and low pressure portion of the ultrasound wave. If the ultrasound pressure is high enough, bubbles will suddenly violently collapse, resulting in impulsive pressures (pressure waves and rapid increase in temperature). These effects are associated with the production of high velocity liquid jets [95, 96].

In an in vivo myocardial transfection by acoustic destruction of gene-laden microbubbles [97], a recombinant adenoviral vector containing the reporter gene -galactosidase was combined with albumin microbubbles and administered intravenously in rats. Transfection occurred in myocardial tissue exposed to high power ultrasound whereas transfection did not occur in the absence of ultrasound or when tissue was exposed to ultrasound and the adenoviral vector without microbubbles. In order to avoid the potentially serious immunogenic and other adverse effects of viral vectors, work has also been done using naked plasmid DNA with microbubbles [98]. Lower ultrasound frequency potentiated higher gene transfection. Gene delivery at the anterior part was higher than at the posterior due to the attenuation of microbubbles and tissues [99]. Combining a catheter-based adenoviral vector delivery system with microbubbles could enhance the efficiency of semi-invasive gene delivery [100].

Ultrasound can enhance cationic lipid-mediated gene transfer to primary tumours following systemic administration [101]. With the addition of plasmid-loaded microbubbles, vascular gene transfer of phosphomimetic endothelial nitric oxide synthase (S1177D) improves vasoreactivity [102].

Microbubble ultrasound has also been used to deliver genes into mouse skeletal muscles [103, 104]. Microbubble ultrasound was used to augment naked plasmid DNA delivery by direct injection into mouse skeletal muscle in vivo [103], in both young (4 week) and older (6 month) mice. It was observed that the albumin-coated microbubble, Optison, significantly improves the transfection efficiency even in the absence of ultrasound. The increase in transgene expression is age related as Optison improves transgene expression less efficiently in older mice than in younger mice. More importantly, Optison markedly reduces muscle damage associated with naked plasmid DNA and the presence of cationic polymer PEI 25000. Ultrasound at moderate power combined with Optison, increased transfection efficiency in older, but not in young, mice. The safe clinical use of microbubbles and therapeutic ultrasound and particularly, the protective effect of the microbubbles against tissue damage provide a highly promising approach for gene delivery in muscle in vivo.

Microbubble ultrasound has been used to transfect nuclear factor B-decoy oligodeoxynucleotides into the donor kidney and prevent acute rejection and prolong graft survival [105]. The kidney was removed and exposed by 2.5 MHz ultrasound at 2.5 W cm^–2 for 1 min and with 10% Optison and transplanted.

	Summary

Top Abstract Introduction Applications to molecular... Applications to molecular... Applications to molecular... Applications to molecular... Summary References

 
The further development of molecular or cellular imaging with ultrasound will involve development of acoustic microscopy made possible by new high frequency transducers and electronics, the expansion of targeted diseases states, improvements in technology for ligand attachment to microbubbles, characterization of the acoustic behaviour of targeted contrast agents, development of better methods for imaging targeted ultrasound agents and optimization of ultrasound-mediated gene and drug delivery.

	Acknowledgments

 
The authors wish to thank Prof. David Cosgrove, Imperial College and Hammersmith Hospital, for helpful comments and advice.

	References

Top Abstract Introduction Applications to molecular... Applications to molecular... Applications to molecular... Applications to molecular... Summary References

 

1. Wells PNT. Physics and engineering: milestones in medicine. Med Eng Phys 2001;23:147–53.[CrossRef][Medline]
2. Blomley MJK, Cooke JC, Unger EC, Monaghan MJ, Cosgrove DO. Science, medicine, and the future - microbubble contrast agents: a new era in ultrasound. Br Med J 2001;322:1222–5.[Free Full Text]
3. Blomley MJK, Sidhu PS, Cosgrove DO, Albrecht T, Harvey CJ, Heckemann RA, et al. Do different types of liver lesions differ in their uptake of the microbubble contrast agent SHU 508A in the late liver phase? Early experience. Radiology 2001;220:661–7.[Abstract/Free Full Text]
4. Schwarz KQ, Bezante GP, Chen XC, Schlief R. Quantitative echo contrast concentration measurement by doppler sonography. Ultrasound Med Biol 1993;19:289–97.[CrossRef][Medline]
5. Wei K, Jayaweera AR, Firoozan S, Linka A, Skyba DM, Kaul S. Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion. Circulation 1998;97:473–83.[Abstract/Free Full Text]
6. Seidel G, Claassen L, Meyer K, Vidal-Langwasser M. Evaluation of blood flow in the cerebral microcirculation: analysis of the refill kinetics during ultrasound contrast agent infusion. Ultrasound Med Biol 2001;27:1059–64.[CrossRef][Medline]
7. Mor-Avi V, Caiani EG, Collins KA, Korcarz CE, Bednarz JE, Lang RM. Combined assessment of myocardial perfusion and regional left ventricular function by analysis of contrast-enhanced power modulation images. Circulation 2001;104:352–7.[Abstract/Free Full Text]
8. Turnbull DH, Bloomfield TS, Baldwin HS, Foster FS, Joyner AL. Ultrasound backscatter microscope analysis of early mouse embryonic brain-development. Proc Natl Acad Sci USA 1995;92:2239–43.[Abstract/Free Full Text]
9. Aristizabal O, Christopher DA, Foster FS, Turnbull DH. 40-MHz echocardiography scanner for cardiovascular assessment of mouse embryos. Ultrasound Med Biol 1998;24:1407–17.[CrossRef][Medline]
10. Srinivasan S, Baldwin HS, Aristizabal O, Kwee L, Labow M, Artman M, et al. Noninvasive, in utero imaging of mouse embryonic heart development with 40-MHz echocardiography. Circulation 1998;98:912–8.[Abstract/Free Full Text]
11. Zhou YQ, Foster FS, Qu DW, Zhang M, Harasiewicz KA, Adamson SL. Applications for multifrequency ultrasound biomicroscopy in mice from implantation to adulthood. Physiol Genomics 2002;10:113–26.[Abstract/Free Full Text]
12. Turnbull DH, Ramsay JA, Shivji GS, Bloomfield TS, From L, Sauder DN, et al. Ultrasound backscatter microscope analysis of mouse melanoma progression. Ultrasound Med Biol 1996;22:845–53.[CrossRef][Medline]
13. Olsson M, Campbell K, Turnbull DH. Specification of mouse telencephalic and mid-hindbrain progenitors following heterotopic ultrasound-guided embryonic transplantation. Neuron 1997;19:761–72.[CrossRef][Medline]
14. Liu AM, Joyner AL, Turnbull DH. Alteration of limb and brain patterning in early mouse embryos by ultrasound-guided injection of Shh-expressing cells. Mech Dev 1998;75:107–15.[CrossRef][Medline]
15. Weiner HL, Bakst R, Hurlbert MS, Ruggiero J, Ahn E, Lee WS, et al. Induction of medulloblastomas in mice by Sonic hedgehog, independent of Gli1. Cancer Res 2002;62:6385–9.[Abstract/Free Full Text]
16. Gaiano N, Kohtz JD, Turnbull DH, Fishell G. A method for rapid gain-of-function studies in the mouse embryonic nervous system. Nat Neurosci 1999;2:812–9.[CrossRef][Medline]
17. Goertz DE, Yu JL, Kerbel RS, Burns PN, Foster FS. High-frequency Doppler ultrasound monitors the effects of antivascular therapy on tumor blood flow. Cancer Res 2002;62:6371–5.[Abstract/Free Full Text]
18. Christopher DA, Burns PN, Starkoski BG, Foster FS. A high-frequency pulsed-wave doppler ultrasound system for the detection and imaging of blood flow in the microcirculation. Ultrasound Med Biol 1997;23:997–1015.[CrossRef][Medline]
19. Berube LR, Harasiewicz K, Foster FS, Dobrowsky E, Sherar MD, Rauth AM. Use of a high-frequency ultrasound microscope to image the action of 2-nitroimidazoles in multicellular spheroids. Br J Cancer 1992;65:633–40.[Medline]
20. Sherar MD, Noss MB, Foster FS. Ultrasound backscatter microscopy images the internal structure of living tumor spheroids. Nature 1987;330:493–5.[CrossRef][Medline]
21. Czarnota GJ, Kolios MC, Vaziri H, Benchimol S, Ottensmeyer FP, Sherar MD, et al. Ultrasonic biomicroscopy of viable, dead and apoptotic cells. Ultrasound Med Biol 1997;23:961–5.[CrossRef][Medline]
22. Czarnota GJ, Kolios MC, Abraham J, Portnoy M, Ottensmeyer FP, Hunt JW, et al. Ultrasound imaging of apoptosis: high-resolution non-invasive monitoring of programmed cell death in vitro, in situ and in vivo. Br J Cancer 1999;81:520–7.[CrossRef][Medline]
23. Hunt JW, Worthington AE, Xuan A, Kolios MC, Czarnota GJ, Sherar MD. A model based upon pseudo regular spacing of cells combined with the randomisation of the nuclei can explain the significant changes in high-frequency ultrasound signals during apoptosis. Ultrasound Med Biol 2002;28:217–26.[CrossRef][Medline]
24. Bereiter-Hahn J, Luers H. The role of elasticity in the motile behaviour of cells. NATO ASI ser. Ser H Cell Biol 1994;84:181–230.
25. Bereiter-Hahn J, Luers H. Subcellular tension fields, mechanical resistance of the lamella front related to the direction of locomotion. Cell Biochem Biophys 1998;29:242–62.
26. Bereiter-Hahn J. Probing biological cells and tissues with acoustic microscopy. Adv Acoustic Microsc 1995;1:79–15.
27. Sarvazyan AP, Scovoroda A, Vucelic D. Utilisation of surface acoustic waves and shear acoustic properties for imaging and tissue characterisation. In: Ermert H, Harjes HP, editors. Acoustical imaging. New York: Plenum Press, 1992:463–8.
28. Sasaki H, Saijo Y, Tanaka M, Okawai H, Terasawa Y, Yambe T, et al. Influence of tissue preparation on the high-frequency acoustic properties of normal kidney tissue. Ultrasound Med Biol 1996;22:1261–5.[CrossRef][Medline]
29. Kundu T, Bereiter-Hahn J, Karl I. Cell property determination from the acoustic microscope generated voltage versus frequency curves. Biophys J 2000;78:2270–9.[Abstract/Free Full Text]
30. Hildebrand JA, Rugar D, Johnston RN, Quate CF. Acoustic microscopy of living cells. Proc Natl Acad Sci USA Biol Sci 1981;78:1656–60.[CrossRef]
31. Hildebrand JA, Rugar D. Measurement of cellular elastic properties by acoustic microscopy. J Microsc Oxf 1984;134:245–60.
32. Litniewski J, Bereiter-Hahn J. Measurements of cells in culture by scanning acoustic microscopy. J Microsc Oxf 1990;158:95–107.
33. Acoustic velocity determination in cytoplasm by V(z) shift. In: Ermert H, Harjes HP, editors. Acoustical Imaging. New York: Plenum Press, 1992:535–8.
34. Kundu T, Bereiter-Hahn J, Hillmann K. Measuring elastic properties of cells by evaluation of scanning acoustic microscopy V(Z) values using simplex algorithm. Biophys J 1991;59:1194–207.
35. Kundu T, Bereiter-Hahn J, Hillman K. Calculating acoustical properties of cells - influence of surface-topography, liquid layer between cell and substrate. J Acoust Soc Am 1992;91:3008–17.[CrossRef][Medline]
36. Barr RJ, White GM, Jones JP, Shaw LB, Ross PA. Scanning acoustic microscopy of neoplastic and inflammatory cutaneous tissue specimens. J Invest Dermatol 1991;96:38–42.[CrossRef][Medline]
37. Jones JP. Applications of acoustical microscopy in dermatology. In: Dunn F, editor. Ultrasonic tissue characterization. New York: Springer-Verlag, 1996:201–12.
38. Saijo Y, Tanaka M, Okawai H, Dunn F. The ultrasonic properties of gastric-cancer tissues obtained with a scanning acoustic microscope system. Ultrasound Med Biol 1991;17:709–14.[CrossRef][Medline]
39. Rubin JM, Adler RS, Fowlkes JB, Spratt S, Pallister JE, Chen JF, et al. Fractional moving blood-volume - estimation with power Doppler US. Radiology 1995;197:183–90.[Abstract/Free Full Text]
40. Peters-Engl C, Medl M, Mirau M, Wanner C, Bilgi S, Sevelda P, et al. Color-coded and spectral Doppler flow in breast carcinomas - relationship with the tumor microvasculature. Breast Cancer Res Treat 1998;47:83–9.[CrossRef][Medline]
41. Gee MS, Saunders HM, Lee JC, Sanzo JF, Jenkins WT, Evans SM, et al. Doppler ultrasound imaging detects changes in tumor perfusion during antivascular therapy associated with vascular anatomic alterations. Cancer Res 2001;61:2974–82.[Abstract/Free Full Text]
42. Kedar RP, Cosgrove D, McCready VR, Bamber JC, Carter ER. Microbubble contrast agent for color Doppler US: effect on breast masses. Work in progress Radiology 1996;198:679–86.
43. Halpern EJ, Rosenberg M, Gomella LG. Prostate cancer: contrast-enhanced US for detection. Radiology 2001;219:219–25.[Abstract/Free Full Text]
44. Bertolotto M, Dalla Palma L, Quaia E, Locatelli M. Characterization of unifocal liver lesions with pulse inversion harmonic imaging after Levovist injection: preliminary results. Eur Radiol 2000;10:1369–76.[CrossRef][Medline]
45. Kim TK, Choi BI, Han JK, Hong HS, Park SH, Moon SG. Hepatic tumors: contrast agent-enhancement patterns with pulse-inversion harmonic US. Radiology 2000;216:411–7.[Abstract/Free Full Text]
46. Blomley MJK, Albrecht H, Cosgrove PO, Jayaram V, Eckersley RJ, Patel N, et al. Liver vascular transit time analyzed with dynamic hepatic venography with bolus injections of an US contrast agent: early experience in seven patients with metastases. Radiology 1998;209:862–6.[Abstract/Free Full Text]
47. Albrecht T, Blomley MJK, Cosgrove DO, Taylor-Robinson SD, Jayaram V, Eckersley R, et al. Non-invasive diagnosis of hepatic cirrhosis by transit-time analysis of an ultrasound contrast agent. Lancet 1999;353:1579–83.[CrossRef][Medline]
48. Blomley MJK, Lim AKP, Harvey CJ, Patel N, Eckersley RJ, Basilico R, et al. Liver microbubble transit time compared with histology and Child-Pugh score in diffuse liver disease: a cross sectional study. Gut 2003;52:1188–93.[Abstract/Free Full Text]
49. Harvey CJ, Blomley MJ, Cosgrove DO, Eckersley RJ, Heckemann RA, Butler-Barnes JA. Can liver vascular transit time measured with bolus injections of the microbubble Levovist predict the presence of occult metastases in colorectal cancer? Radiology 2000;217:816.
50. Lindner JR, Coggins MP, Kaul S, Klibanov AL, Brandenburger GH, Ley K. Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin- and complement-mediated adherence to activated leukocytes. Circulation 2000;101:668–75.[Abstract/Free Full Text]
51. Lanza GM, Wickline SA. Targeted ultrasonic contrast agents for molecular imaging and therapy. Prog Cardiovasc Dis 2001;44:13–31.[CrossRef][Medline]
52. Kono Y, Steinbach GC, Peterson T, Schmid-Schonbein GW, Mattrey RF. Mechanism of parenchymal enhancement of the liver with a microbubble-based US contrast medium: an intravital microscopy study in rats. Radiology 2002;224:253–7.[Abstract/Free Full Text]
53. Forsberg F, Goldberg BB, Liu JB, Merton DA, Rawool NM, Shi WT. Tissue-specific US contrast agent for evaluation of hepatic and splenic parenchyma. Radiology 1999;210:125–32.[Abstract/Free Full Text]
54. Villanueva FS, Jankowski RJ, Manaugh C, Wagner WR. Albumin microbubble adherence to human coronary endothelium: implications for assessment of endothelial function using myocardial contrast echocardiography. J Am Coll Cardiol 1997;30:689–93.[Abstract]
55. Lindner JR, Dayton PA, Coggins MP, Ley K, Song J, Ferrara K, et al. Noninvasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles. Circulation 2000;102:531–8.[Abstract/Free Full Text]
56. Lindner JR, Song J, Xu F, Klibanov AL, Singbartl K, Ley K, et al. Noninvasive ultrasound imaging of inflammation using microbubbles targeted to activated leukocytes. Circulation 2000;102:2745–50.[Abstract/Free Full Text]
57. Walzog B, Schuppan D, Heimpel C, Hafezimoghadam A, Gaehtgens P, Ley K. The leukocyte integrin Mac-1 (Cd11b/Cd18) contributes to binding of human granulocytes to collagen. Exp Cell Res 1995;218:28–38.[CrossRef][Medline]
58. Davis GE. The Mac-1 and P150,95 Beta-2 integrins bind denatured proteins to mediate leukocyte cell substrate adhesion. Exp Cell Res 1992;200:242–52.[CrossRef][Medline]
59. Dayton PA, Chomas JE, Lum AFH, Allen JS, Lindner JR, Simon SI, et al. Optical and acoustical dynamics of microbubble contrast agents inside neutrophils. Biophys J 2001;80:1547–56.[Abstract/Free Full Text]
60. Villanueva FS, Jankowski RJ, Klibanov S, Pina ML, Alber SM, Watkins SC, et al. Microbubbles targeted to intercellular adhesion molecule-1 bind to activated coronary artery endothelial cells. Circulation 1998;98:1–5.[Abstract/Free Full Text]
61. Lindner JR, Song J, Christiansen J, Klibanov AL, Xu F, Ley K. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation 2001;104:2107–12.[Abstract/Free Full Text]
62. Barbarese E, Ho SY, Darrigo JS, Simon RH. Internalization of microbubbles by tumor-cells in-vivo and in-vitro. J Neurooncol 1995;26:25–34.[CrossRef][Medline]
63. Ho SY, Barbarese E, Darrigo JS, SmithSlatas C, Simon RH. Evaluation of lipid-coated microbubbles as a delivery vehicle for taxol in brain tumor therapy. Neurosurgery 1997;40:1260–6.[CrossRef][Medline]
64. Wakefield AE, Ho SY, Li XG, D'Arrigo JS, Simon RH. The use of lipid-coated microbubbles as a delivery agent of 7 beta-hydroxycholesterol in a radiofrequency lesion in the rat brain. Neurosurgery 1998;42:592–8.[CrossRef][Medline]
65. Ellegala DB, Poi HL, Carpenter JE, Klibanov AL, Kaul S, Shaffrey ME, et al. Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to alpha(v)beta(3). Circulation 2003;108:336–41.[Abstract/Free Full Text]
66. Patel D, Dayton P, Gut J, Wisner E, Ferrara KW. Optical and acoustical interrogation of submicron contrast agents. IEEE Trans Ultrason Ferroelectr Freq Control 2002;49:1641–51.[CrossRef][Medline]
67. Unger EC, McCreery TP, Sweitzer RH, Shen DK, Wu GL. In vitro studies of a new thrombus-specific ultrasound contrast agent. Am J Cardiol 1998;81:58G–61G.[CrossRef][Medline]
68. Wright WH, McCreery TP, Krupinski EA, Lund PJ, Smyth SH, Baker MR, et al. Evaluation of new thrombus-specific ultrasound contrast agent. Acad Radiol 1998;5:S240–S242.
69. Takeuchi M, Ogunyankin K, Pandian NG, McCreery TP, Sweitzer RH, Caldwell VE, et al. Enhanced visualization of intravascular and left atrial appendage thrombus with the use of a thrombus-targeting ultrasonographic contrast agent (MRX-408A1): in vivo experimental echocardiographic studies. J Am Soc Echocardiogr 1999;12:1015–21.[CrossRef][Medline]
70. Schumann PA, Christiansen JP, Quigley RM, McCreery TP, Sweitzer RH, Unger EC, et al. Targeted-microbubble binding selectively to GPIIb IIIa receptors of platelet thrombi. Invest Radiol 2002;37:587–93.[CrossRef][Medline]
71. Ruoslahti E. RGD and other recognition sequences for integrins. Ann Rev Cell Dev Biol 1996;12:697–715.[CrossRef][Medline]
72. Wu YQ, Unger EC, McCreery TP, Sweitzer RH, Shen DK, Wu GL, et al. Binding and lysing of blood clots using MRX-408. Invest Radiol 1998;33:880–5.[CrossRef][Medline]
73. Alkan-Onyuksel H, Demos SM, Lanza GM, Vonesh MJ, Klegerman ME, Kane BJ, et al. Development of inherently echogenic liposomes as an ultrasonic contrast agent. J Pharm Sci 1996;85:486–90.[CrossRef][Medline]
74. Demos SM, Onyuksel H, Gilbert J, Roth SI, Kane B, Jungblut P, et al. In vitro targeting of antibody-conjugated echogenic liposomes for site-specific ultrasonic image enhancement. J Pharm Sci 1997;86:167–71.[CrossRef][Medline]
75. Demos SM, Dagar S, Klegerman M, Nagaraj A, McPherson DD, Onyuksel H. In vitro targeting of acoustically reflective immunoliposomes to fibrin under various flow conditions. J Drug Target 1998;5:507–18.[Medline]
76. Demos SM, Alkan-Onyuksel H, Kane BJ, Ramani K, Nagaraj A, Greene R, et al. In vivo targeting of acoustically reflective liposomes for intravascular and transvascular ultrasonic enhancement. J Am Coll Cardiol 1999;33:867–75.[Abstract/Free Full Text]
77. Huang SL, Hamilton AJ, Pozharski E, Nagaraj A, Klegerman ME, McPherson DD, et al. Physical correlates of the ultrasonic reflectivity of lipid dispersions suitable as diagnostic contrast agents. Ultrasound Med Biol 2002;28:339–48.[CrossRef][Medline]
78. Lanza GM, Wallace KD, Scott MJ, Cacheris WP, Abendschein DR, Christy DH, et al. A novel site-targeted ultrasonic contrast agent with broad biomedical application. Circulation 1996;94:3334–40.[Abstract/Free Full Text]
79. Lanza GM, Wallace KD, Fischer SE, Christy DH, Scott MJ, Trousil RL, et al. High-frequency ultrasonic detection of thrombi with a targeted contrast system. Ultrasound Med Biol 1997;23:863–70.[CrossRef][Medline]
80. Lanza GM, Abendschein DR, Hall CS, Scott MJ, Scherrer DE, Houseman A, et al. In vivo molecular imaging of stretch-induced tissue factor in carotid arteries with ligand-targeted nanoparticles. J Am Soc Echocardiogr 2000;13:608–14.[CrossRef][Medline]
81. Lanza GM, Abendschein DR, Hall CS, Marsh JN, Scott MJ, Scherrer DE, et al. Molecular imaging of stretch-induced tissue factor expression in carotid arteries with intravascular ultrasound. Invest Radiol 2000;35:227–34.[CrossRef][Medline]
82. Lanza GM, Trousil RL, Wallace KD, Rose JH, Hall CS, Scott MJ, et al. In vitro characterization of a novel, tissue-targeted ultrasonic contrast system with acoustic microscopy. J Acoust Soc Am 1998;104:3665–72.[CrossRef][Medline]
83. Hall CS, Marsh JN, Scott MJ, Gaffney PJ, Wickline SA, Lanza GM. Time evolution of enhanced ultrasonic reflection using a fibrin-targeted nanoparticulate contrast agent. J Acoust Soc Am 2000;108:3049–57.[CrossRef][Medline]
84. Hall CS, Marsh JN, Scott MJ, Gaffney PJ, Wickline SA, Lanza GM. Temperature dependence of ultrasonic enhancement with a site-targeted contrast agent. J Acoust Soc Am 2001;110:1677–84.[CrossRef][Medline]
85. Hall CS, Lanza GM, Rose JH, Kaufmann RJ, Fuhrhop RW, Handley SH, et al. Experimental determination of phase velocity of perfluorocarbons: applications to targeted contrast agents. IEEE Trans Ultrason Ferroelectr Freq Control 2000;47:75–84.[Medline]
86. Marsh JN, Hall CS, Scott MJ, Fuhrhop RW, Gaffney PJ, Wickline SA, et al. Improvements in the ultrasonic contrast of targeted perfluorocarbon nanoparticles using an acoustic transmission line model. IEEE Trans Ultrason Ferroelectr Freq Control 2002;49:29–38.[CrossRef][Medline]
87. Flacke S, Fischer S, Scott MJ, Fuhrhop RJ, Allen JS, McLean M, et al. Novel MRI contrast agent for molecular imaging of fibrin implications for detecting vulnerable plaques. Circulation 2001;104:1280–5.[Abstract/Free Full Text]
88. Fechheimer M, Boylan JF, Parker S, Sisken JE, Patel GL, Zimmer SG. Transfection of mammalian-cells with plasmid DNA by scrape loading and sonication loading. Proc Natl Acad Sci USA 1987;84:8463–7.[Abstract/Free Full Text]
89. Miller DL, Bao SP, Morris JE. Sonoporation of cultured cells in the rotating tube exposure system. Ultrasound Med Biol 1999;25:143–9.[CrossRef][Medline]
90. Fechheimer M, Denny C, Murphy RF, Taylor DL. Measurement of cytoplasmic Ph in dictyostelium discoideum by using a new method for introducing macromolecules into living cells. Eur J Cell Biol 1986;40:242–7.[Medline]
91. Guzman HR, Nguyen DX, Khan S, Prausnitz MR. Ultrasound-mediated disruption of cell membranes. I. Quantification of molecular uptake and cell viability. J Acoust Soc Am 2001;110:588–96.[CrossRef][Medline]
92. Guzman HR, Nguyen DX, Khan S, Prausnitz MR. Ultrasound-mediated disruption of cell membranes. II. Heterogeneous effects on cells. J Acoust Soc Am 2001;110:597–606.[CrossRef][Medline]
93. Bao SP, Thrall BD, Miller DL. Transfection of a reporter plasmid into cultured cells by sonoporation in vitro. Ultrasound Med Biol 1997;23:953–9.[CrossRef][Medline]
94. Harrison GH, BalcerKubiczek EK, Gutierrez PL. In vitro mechanisms of chemopotentiation by tone-burst ultrasound. Ultrasound Med Biol 1996;22:355–62.[CrossRef][Medline]
95. Kodama T, Takayama K. Dynamic behavior of bubbles during extracorporeal shock-wave lithotripsy. Ultrasound Med Biol 1998;24:723–38.[CrossRef][Medline]
96. Kodama T, Tomita Y. Cavitation bubble behavior and bubble-shock wave interaction near a gelatin surface as a study of in vivo bubble dynamics. Appl Phys B-Lasers Opt 2000;70:139–49.[CrossRef]
97. Shohet RV, Chen SY, Zhou YT, Wang ZW, Meidell RS, Unger TH, et al. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation 2000;101:2554–6.[Abstract/Free Full Text]
98. Chen SY, Shohet RV, Frenkel P, Mayer S, Unger RH, Grayburn PA. Successful expression of plasmid DNA in rat myocardium by ultrasound-targeted microbubble destruction. J Am Coll Cardiol 2001;37:407A.
99. Chen SY, Shohet RV, Bekeredjian R, Frenkel P, Grayburn PA. Optimization of ultrasound parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by ultrasound-targeted microbubble destruction. J Am Coll Cardiol 2003;42:301–8.[Abstract/Free Full Text]
100. Beeri R, Guerrero JL, Supple G, Sullivan S, Levine RA, Hajjar RJ. New efficient catheter-based system for myocardial gene delivery. Circulation 2002;106:1756–9.[Abstract/Free Full Text]
101. Anwer K, Kao G, Proctor B, Anscombe I, Florack V, Earls R, et al. Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration. Gene Ther 2000;7:1833–9.[CrossRef][Medline]
102. Teupe C, Richter S, Fisslthaler B, Randriamboavonjy V, Ihling C, Fleming I, et al. Vascular gene transfer of phosphomimetic endothelial nitric oxide synthase (S1177D) using ultrasound-enhanced destruction of plasmid-loaded microbubbles improves vasoreactivity. Circulation 2002;105:1104–9.[Abstract/Free Full Text]
103. Lu QL, Liang HD, Partridge T, Blomley MJK. Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage. Gene Ther 2003;10:396–405.[CrossRef][Medline]
104. Taniyama Y, Tachibana K, Hiraoka K, Aoki M, Yamamoto S, Matsumoto K, et al. Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Ther 2002;9:372–80.[CrossRef][Medline]
105. Azuma H, Tomita N, Kaneda Y, Koike H, Ogihara T, Katsuoka Y, et al. Transfection of NF kappa B-decoy oligodeoxynucleotides using efficient ultrasound-mediated gene transfer into donor kidneys prolonged survival of rat renal allografts. Gene Ther 2003;10:415–25.[CrossRef][Medline]

This article has been cited by other articles:

W. Cai and X. Chen Multimodality Molecular Imagi