Phase shift variance imaging - a new technique for destructive microbubble imaging.

The detection of microbubble contrast agents with ultrasound imaging techniques is the subject of ongoing research. Commonly, the nonlinear response of the agent is employed for detection. The performance of these techniques is, however, affected by nonlinear sound propagation. As an alternative, the change in echo response resulting from microbubble destruction can be employed to detect the agent. In this work, we propose a novel criterion for microbubble destruction detection that allows the rejection of tissue at a defined significance level even for highly echogenic structures in the presence of nonlinear propagation. Most clinical systems provide the hardware requirements for acquisitions consisting of multiple pulses transmitted at the same position, as used in Doppler imaging. Therefore, we develop a processing strategy that distinguishes contrast agent from other stationary or moving structures using these sequences. The proposed criterion is based on the variance of the phase shift of consecutive echoes in the sequence, which, in addition to tissue rejection, permits the distinction of motion from agent disruption. Phantom experiments are conducted to show the validity of the criterion and demonstrate the performance of the new method for contrast detection. Each detection series consists of 20 identical pulses at 9.5 MHz (4.7 MPa peak negative pressure) transmitted at a pulse repetition frequency of 5 kHz. The sequence is applied to phantoms under varied motion and flow conditions. As a first step toward molecular imaging, the technique is applied to microbubbles targeted to vascular endothelial growth factor receptor 2 (VEGFR2) in vitro. The results show a uniform rejection of the background signal while maintaining a contrast enhancement by more than 40 dB. The area under the receiver operating characteristics (ROC) curve is used as the performance metric for the separation of contrast agent and tissue signals, and values larger than 97% demonstrate that an excellent separation was achieved.

Influence of shell composition on the resonance frequency of microbubble contrast agents.

The effect of variations in microbubble shell composition on microbubble resonance frequency is revealed through experiment. These variations are achieved by altering the mole fraction and molecular weight of functionalized polyethylene glycol (PEG) in the microbubble phospholipid monolayer shell and measuring the microbubble resonance frequency. The resonance frequency is measured via a chirp pulse and identified as the frequency at which the pressure amplitude loss of the ultrasound wave is the greatest as a result of passing through a population of microbubbles. For the shell compositions used herein, we find that PEG molecular weight has little to no influence on resonance frequency at an overall PEG mole fraction (0.01) corresponding to a mushroom regime and influences the resonance frequency markedly at overall PEG mole fractions (0.050-0.100) corresponding to a brush regime. Specifically, the measured resonance frequency was found to be 8.4, 4.9, 3.3 and 1.4 MHz at PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.075. At an overall PEG mole fraction of just 0.01, on the other hand, resonance frequency exhibited no systematic variation, with values ranging from 5.7 to 4.9 MHz. Experimental results were analyzed using the Sarkar bubble dynamics model. With the dilatational viscosity held constant (10(-8) N·s/m) and the elastic modulus used as a fitting parameter, model fits to the pressure amplitude loss data resulted in elastic modulus values of 2.2, 2.4, 1.6 and 1.8 N/m for PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.010 and 4.2, 1.4, 0.5 and 0.0 N/m, respectively, at an overall PEG mole fraction of 0.075. These results are consistent with theory, which predicts that the elastic modulus is constant in the mushroom regime and decreases with PEG molecular weight to the inverse 3/5 power in the brush regime. Additionally, these results are consistent with inertial cavitation studies, which revealed that increasing PEG molecular weight has little to no effect on inethe rtial cavitation threshold in the mushroom regime, but that increasing PEG molecular weight decreases inertial cavitation markedly in the brush regime. We conclude that the design and synthesis of microbubbles with a prescribed resonance frequency is attainable by tuning PEG composition and molecular weight.

Targeted ultrasound imaging of cancer: an emerging technology on its way to clinics.

Ultrasound is one of the workhorses in clinical cancer diagnosis. In particular, it is routinely used to characterize lesions in liver, urogenital tract, head and neck and soft tissues. During the last years image quality steadily improved, which, among others, can be attributed to the development of harmonic image analysis. Microbubbles were introduced as intravascular contrast agents and can be detected with superb sensitivity and specificity using contrast specific imaging modes. By aid of these unspecific contrast agents tissues can be characterised regarding their vascularity. Antibodies, peptides and other targeting moieties were bound to microbubbles to target sites of angiogenesis and inflammation intending to get more disease-specific information. Indeed, many preclinical studies proved the high potential of targeted ultrasound imaging to better characterize tumors and to more sensitively monitor therapy response. Recently, first targeted microbubbles had been developed that meet the pharmacological demands of a clinical contrast agent. This review articles gives an overview on the history and current status of targeted ultrasound imaging of cancer. Different imaging concepts and contrast agent designs are introduced ranging from the use of experimental nanodroplets to agents undergoing clinical evaluation. Although it is clear that targeted ultrasound imaging works reliably, its broad acceptance is hindered by the user dependency of ultrasound imaging in general. Automated 3D-scanning techniques-like being used for breast diagnosis - and novel 3D transducers will help to make this fascinating method clinical reality.

Evaluation of high frequency ultrasound methods and contrast agents for characterising tumor response to anti-angiogenic treatment.

PURPOSE: To compare non-enhanced and contrast-enhanced high-frequency 3D Doppler ultrasound with contrast-enhanced 2D and 3D B-mode imaging for assessing tumor vascularity during antiangiogenic treatment using soft-shell and hard-shell microbubbles. MATERIALS AND METHODS: Antiangiogenic therapy effects (SU11248) on vascularity of subcutaneous epidermoid-carcinoma xenografts (A431) in female CD1 nude mice were investigated longitudinally using non-enhanced and contrast-enhanced 3D Doppler at 25 MHz. Additionally, contrast-enhanced 2D and 3D B-mode scans were performed by injecting hard-shell (poly-butyl-cyanoacrylate-based) and soft-shell (phospholipid-based) microbubbles. Suitability of both contrast agents for high frequency imaging and the sensitivity of the different ultrasound methods to assess early antiangiogenic therapy effects were investigated. Ultrasound data were validated by immunohistology. RESULTS: Hard-shell microbubbles induced higher signal intensity changes in tumors than soft-shell microbubbles in 2D B-mode measurements (424 ± 7 vs. 169 ± 8 A.U.; p<0.01). In 3D measurements, signals of soft-shell microbubbles were hardly above the background (5.48 ± 4.57 vs. 3.86 ± 2.92 A.U.), while signals from hard-shell microbubbles were sufficiently high (30.5 ± 8.06 A.U). Using hard-shell microbubbles 2D and 3D B-mode imaging depicted a significant decrease in tumor vascularity during antiangiogenic therapy from day 1 on. Using soft-shell microbubbles significant therapy effects were observed at day 4 after therapy in 2D B-mode imaging but could not be detected in the 3D mode. With non-enhanced and contrast-enhanced Doppler imaging significant differences between treated and untreated tumors were found from day 2 on. CONCLUSION: Hard-shell microbubble-enhanced 2D and 3D B-mode ultrasound achieved highest sensitivity for assessing therapy effects on tumor vascularisation and were superior to B-mode ultrasound with soft-shell microbubbles and to Doppler imaging.

Advanced characterization and refinement of poly N-butyl cyanoacrylate microbubbles for ultrasound imaging.

We aimed to develop and characterize poly n-butylcyanoacrylate (PBCA) microbubbles (MBs) with a narrow size distribution. MBs were synthesized by established emulsion polymerization techniques, size-isolated by centrifugation and functionalized for molecular imaging by coating their surface with streptavidin. The physical and acoustic properties of the parent solution, different-size isolated populations and functionalized MBs were measured and compared. As expected from negative zeta potentials at pH 7, cryo scanning electron microscopy showed no aggregates. In phantoms MBs were destructible at high mechanical indices and showed a frequency-dependent attenuation and backscattering. The MBs were stable in solution for more than 14 weeks and could be lyophilized without major damage. However, for injection, small needle diameters and high injection rates are shown to be critical because both lead to MB destruction. In summary, when being handled correctly, size-isolated PBCA MBs are promising candidates for preclinical functional and molecular ultrasound imaging.

Molecular and functional ultrasound imaging in differently aggressive breast cancer xenografts using two novel ultrasound contrast agents (BR55 and BR38).

OBJECTIVES: To characterise clinically translatable long-circulating (BR38) and VEGFR2-targeted (BR55) microbubbles (MB) and to assess their ability to discriminate breast cancer models with different aggressiveness. METHODS: The circulation characteristics of BR38 and BR55 were investigated in healthy mice. The relative blood volume (rBV) of MDA-MB-231 (n = 5) or MCF-7 (n = 6) tumours was determined using BR38. In the same tumours in-vivo binding specificity of BR55 was tested and VEGFR2 expression assessed. Data validation included quantitative immunohistological analysis. RESULTS: BR38 had a longer blood half-life than BR55 (>600 s vs. 218 s). BR38-enhanced ultrasound showed greater vascularisation in MDA-MB-231 tumours (p = 0.022), which was in line with immunohistology (p = 0.033). In-vivo competitive binding experiments proved the specificity of BR55 to VEGFR2 (p = 0.027). Binding of BR55 was significantly higher in MDA-MB-231 than in MCF-7 tumours (p = 0.049), which corresponded with the VEGFR2 levels found histologically (p = 0.015). However, differences became smaller when normalising the levels of BR55 to the rBV. CONCLUSIONS: BR38 and BR55 are well suited to characterising and distinguishing breast cancers with different angiogenesis and aggressiveness. Long-circulating BR38 MB allow extensive 3-dimensional examinations of larger or several organs. BR55 accumulation faithfully reflects the VEGFR2 status in tumours and depicts even small differences in angiogenesis.

Synergistic effects of sonoporation and taurolidin/TRAIL on apoptosis in human fibrosarcoma.

Sonodynamic therapy, in combination with ultrasound contrast agents, proved to enhance the uptake of chemotherapeutics in malignant cells. HT1080 fibrosarcoma cells were treated in vitro with a combination of ultrasound SonoVue™-microbubbles and taurolidine (TRD) plus tumor necrosis factor related apoptosis inducing ligand (TRAIL). Apoptosis was measured by TdT-mediated dUTP-biotin nick end labelling (TUNEL) assay and fluorescence activated cell sorting (FACS) analysis. Gene expression was analysed by RNA-microarray. The apoptotic effects of TRD and TRAIL on human fibrosarcoma are enhanced by sonodynamic therapy and additional application of contrast agents, such as SonoVue™ by 25%. A broad change in the expression of genes related to apoptotic pathways is observed when ultrasound and microbubbles act synchronously in combination with the chemotherapeutics (e.g. BIRC3, NFKBIA and TNFAIP3). Some of these genes have already been proven to play a role in programmed cell death in human fibrosarcoma (HSPA1A/HSPA1B, APAF1, PAWR, SOCS2) or were associated with sonication induced apoptosis (CD44). Further studies are needed to explore the options of sonodynamic therapy on soft tissue sarcoma and its molecular mechanisms.

A statistical model for the quantification of microbubbles in destructive imaging.

RATIONALE AND OBJECTIVES: Quantification of targeted ultrasound contrast agents allows for the monitoring of endothelial marker expressions on a molecular level. In this study, a statistical correction is provided, which allows for improved precision in estimating the concentration of microbubbles (MBs) from Doppler images. Doppler imaging can be used to display the destruction of single MBs. However, concentrations will generally be too high to distinguish the individual events resulting in an inaccurate microbubble (MB) quantification. Therefore, a mathematical description of destruction events in Doppler images is developed which yields a correction formula for the concentration estimate from the color pixel density. METHODS: The mathematical model is experimentally verified in gelatin phantoms using a high resolution imaging system (Vevo 770) and experimental cyanoacrylate MBs. Sensitive Particle Acoustic Quantification (SPAQ) is used to quantify MB in a defined volume. The SPAQ step size is varied from 32 to 127 µm to demonstrate the validity of the model for high color pixel densities. RESULTS: The corrected acoustic quantification shows the expected linear dependence on the step size and thus the amount of MBs in the images (R = 0.95). At SPAQ step sizes up to 127 µm, a MB concentration of 2.7 × 10 MBs/mL can be quantified. CONCLUSIONS: The results demonstrate the validity of the proposed correction. Quantification results of the SPAQ technique were considerably improved. The resulting formula is readily applied to SPAQ measurements at no additional expense.