Verya Daeichin

Targeted ultrasound contrast agents for ultrasound molecular imaging and therapy

Abstract Ultrasound contrast agents (UCAs) are used routinely in the clinic to enhance contrast in ultrasonography. More recently, UCAs have been functionalised by conjugating ligands to their surface to target specific biomarkers of a disease or a disease process. These targeted UCAs (tUCAs) are used for a wide range of pre-clinical applications including diagnosis, monitoring of drug treatment, and therapy. In this review, recent achievements with tUCAs in the field of molecular imaging, evaluation of therapy, drug delivery, and therapeutic applications are discussed. We present the different coating materials and aspects that have to be considered when manufacturing tUCAs. Next to tUCA design and the choice of ligands for specific biomarkers, additional techniques are discussed that are applied to improve binding of the tUCAs to their target and to quantify the strength of this bond. As imaging techniques rely on the specific behaviour of tUCAs in an ultrasound field, it is crucial to understand the characteristics of both free and adhered tUCAs. To image and quantify the adhered tUCAs, the state-of-the-art techniques used for ultrasound molecular imaging and quantification are presented. This review concludes with the potential of tUCAs for drug delivery and therapeutic applications.

SUBHARMONIC, NON-LINEAR FUNDAMENTAL AND ULTRAHARMONIC IMAGING OF MICROBUBBLE CONTRAST AT HIGH FREQUENCIES

There is increasing use of ultrasound contrast agent in high-frequency ultrasound imaging. However, conventional contrast detection methods perform poorly at high frequencies. We performed systematic in vitro comparisons of subharmonic, non-linear fundamental and ultraharmonic imaging for different depths and ultrasound contrast agent concentrations (Vevo 2100 system with MS250 probe and MicroMarker ultrasound contrast agent, VisualSonics, Toronto, ON, Canada). We investigated 4-, 6- and 10-cycle bursts at three power levels with the following pulse sequences: B-mode, amplitude modulation, pulse inversion and combined pulse inversion/amplitude modulation. The contrast-to-tissue (CTR) and contrast-to-artifact (CAR) ratios were calculated. At a depth of 8 mm, subharmonic pulse-inversion imaging performed the best (CTR = 26 dB, CAR = 18 dB) and at 16 mm, non-linear amplitude modulation imaging was the best contrast imaging method (CTR = 10 dB). Ultraharmonic imaging did not result in acceptable CTRs and CARs. The best candidates from the in vitro study were tested in vivo in chicken embryo and mouse models, and the results were in a good agreement with the in vitro findings.

Unique pumping-out fracturing mechanism of a polymer-shelled contrast agent: an acoustic characterization and optical visualization

This work describes the fracturing mechanism of air-filled microbubbles (MBs) encapsulated by a cross-linked poly(vinyl alcohol) (PVA) shell. The radial oscillation and fracturing events following the ultrasound exposure were visualized with an ultrahigh-speed camera, and backscattered timedomain signals were acquired with the acoustic setup specific for harmonic detection. No evidence of gas emerging from defects in the shell with the arrival of the first insonation burst was found. In optical recordings, more than one shell defect was noted, and the gas core was drained without any sign of air extrusion when several consecutive bursts of 1 MPa amplitude were applied. In acoustic tests, the backscattered peak-to-peak voltage gradually reached its maximum and exponentially decreased when the PVA-based MB suspension was exposed to approximately 20 consecutive bursts arriving at pulse repetition frequencies of 100 and 500 Hz. Taking into account that the PVA shell is porous and possibly contains large air pockets between the cross-linked PVA chains, the aforementioned acoustic behavior might be attributed to pumping gas from these pockets in combination with gas release from the core through shell defects. We refer to this fracturing mechanism as pumping-out behavior, and this behavior could have potential use for the local delivery of therapeutic gases, such as nitric oxide.

Quantification of bound microbubbles in ultrasound molecular imaging

Molecular markers associated with diseases can be visualized and quantified noninvasively with targeted ultrasound contrast agent (t-UCA) consisting of microbubbles (MBs) that can bind to specific molecular targets. Techniques used for quantifying t-UCA assume that all unbound MBs are taken out of the blood pool few minutes after injection and only MBs bound to the molecular markers remain. However, differences in physiology, diseases, and experimental conditions can increase the longevity of unbound MBs. In such conditions, unbound MBs will falsely be quantified as bound MBs. We have developed a novel technique to distinguish and classify bound from unbound MBs. In the post-processing steps, first, tissue motion was compensated using block-matching (BM) techniques. To preserve only stationary contrast signals, a minimum intensity projection (MinIP) or 20th-percentile intensity projection (PerIP) was applied. The after-flash MinIP or PerIP was subtracted from the before-flash MinIP or PerIP. In this way, tissue artifacts in contrast images were suppressed. In the next step, bound MB candidates were detected. Finally, detected objects were tracked to classify the candidates as unbound or bound MBs based on their displacement. This technique was validated in vitro, followed by two in vivo experiments in mice. Tumors (n = 2) and salivary glands of hypercholesterolemic mice (n = 8) were imaged using a commercially available scanner. Boluses of 100 μL of a commercially available t-UCA targeted to angiogenesis markers and untargeted control UCA were injected separately. Our results show considerable reduction in misclassification of unbound MBs as bound ones. Using our method, the ratio of bound MBs in salivary gland for images with targeted UCA versus control UCA was improved by up to two times compared with unprocessed images.

A Broadband Polyvinylidene Difluoride-Based Hydrophone with Integrated Readout Circuit for Intravascular Photoacoustic Imaging

Intravascular photoacoustic (IVPA) imaging can visualize the coronary atherosclerotic plaque composition on the basis of the optical absorption contrast. Most of the photoacoustic (PA) energy of human coronary plaque lipids was found to lie in the frequency band between 2 and 15 MHz requiring a very broadband transducer, especially if a combination with intravascular ultrasound is desired. We have developed a broadband polyvinylidene difluoride (PVDF) transducer (0.6 × 0.6 mm, 52 μm thick) with integrated electronics to match the low capacitance of such a small polyvinylidene difluoride element (<5 pF/mm(2)) with the high capacitive load of the long cable (∼100 pF/m). The new readout circuit provides an output voltage with a sensitivity of about 3.8 μV/Pa at 2.25 MHz. Its response is flat within 10 dB in the range 2 to 15 MHz. The root mean square (rms) output noise level is 259 μV over the entire bandwidth (1-20 MHz), resulting in a minimum detectable pressure of 30 Pa at 2.25 MHz.

Optimization of ultrasound contrast agent for high frequency ultrasound molecular imaging using subharmonic oscillation

Ultrasound molecular imaging visualizes disease biomarkers using targeted ultrasound contrast agents (t-UCA). Different lipids result in UCA with different acoustic behavior. In this study, subharmonic (SH) response of MicroMarker UCA and four homemade UCA with different lipid coatings were compared in vitro with a high frequency ultrasound system (Vevo 2100 with MS250 probe). UCA with different size distribution were made with DSPC (UCA A and C) or DPPC (UCA B and D) as main lipid by sonication or Vialmix. We transmitted long bursts (20-cycle, 30 MHz, 10% power, MS250 probe) with pulse inversion sequence for optimized SH imaging. The effect of the UCA concentration on SH imaging was tested using two concentrations: 8.0×106 and 4.0×105 microbubbles per milliliter. For A, B and D type UCA, attenuation effect is dominant at high concentration. MicroMarker and C type UCA had the highest and most spatially homogeneous distribution of SH response throughout the entire UCA area at high concentration. SH amplitude of MicroMarker dropped about 11 dB when UCA concentration was reduced by a factor of 20. For this low concentration, SH amplitude of B type UCA was 10 dB higher than that for MicroMarker and was homogeneous throughout the UCA area. We showed that for 30 MHz transmit frequency, at low concentration of UCA, DPPC microbubbles have higher SH response than DSPC ones and at high concentration, smaller bubbles (MicroMarker and C) produced the most homogeneous SH responses. Our results suggest that for molecular imaging applications where UCA concentration is low, our B-type and MicroMarker UCA are suitable choices for SH imaging.

Multiline 3D beamforming using micro-beamformed datasets for pediatric transesophageal echocardiography

Until now, no matrix transducer has been realized for 3D transesophageal echocardiography (TEE) in pediatric patients. In 3D TEE with a matrix transducer, the biggest challenges are to connect a large number of elements to a standard ultrasound system, and to achieve a high volume rate (>200 Hz). To address these issues, we have recently developed a prototype miniaturized matrix transducer for pediatric patients with micro-beamforming and a small central transmitter. In this paper we propose two multiline parallel 3D beamforming techniques (µBF25 and µBF169) using the micro-beamformed datasets from 25 and 169 transmit events to achieve volume rates of 300 Hz and 44 Hz, respectively. Both the realizations use angle-weighted combination of the neighboring overlapping sub-volumes to avoid artifacts due to sharp intensity changes introduced by parallel beamforming. In simulation, the image quality in terms of the width of the point spread function (PSF), lateral shift invariance and mean clutter level for volumes produced by µBF25 and µBF169 are similar to the idealized beamforming using a conventional single-line acquisition with a fully-sampled matrix transducer (FS4k, 4225 transmit events). For completeness, we also investigated a 9 transmit-scheme (3  ×  3) that allows even higher frame rates but found worse B-mode image quality with our probe. The simulations were experimentally verified by acquiring the µBF datasets from the prototype using a Verasonics V1 research ultrasound system. For both µBF169 and µBF25, the experimental PSFs were similar to the simulated PSFs, but in the experimental PSFs, the clutter level was ~10 dB higher. Results indicate that the proposed multiline 3D beamforming techniques with the prototype matrix transducer are promising candidates for real-time pediatric 3D TEE.

Optimizing the directivity of piezoelectric matrix transducer elements mounted on an ASIC

Over the last decade, clinical studies show a strong interest in real-time 3D imaging. This calls for ultrasound probes with high-element-count 2D matrix transducer arrays. These may be interfaced to an imaging system using an in-probe Application Specific Integrated Circuit (ASIC) that takes care of signal amplification, element switching, sub-array beamforming, etc. Since the ASIC is made from silicon and is mounted directly behind the transducer elements, it can acoustically be regarded as a rigid plate that can sustain traveling lateral waves. These waves lead to acoustical cross-talk between the elements, and results in extra peaks in the directivity pattern. We propose two solutions to this problem, based on numerical simulations. One approach is to decrease the phase velocity in the silicon by reducing the silicon thickness and absorbing the energy using a proper backing material. Another solution is to disturb the waves inside the silicon plate by sub-dicing the back-side of the ASIC. We conclude that both solutions can be used to improve the directivity pattern.

A front-end ASIC with high-voltage transmit switching and receive digitization for forward-looking intravascular ultrasound

This paper presents a front-end ASIC for forward-looking intravascular ultrasound (IVUS) imaging. The ASIC is intended to be mounted at the tip of a catheter and can interface a total of 80 piezo-electric transducer elements with an imaging systems using only 4 cables, thus significantly reducing the system complexity compared to the prior art. It is capable of switching high-voltage transmit pulses to 16 transmit elements, and capturing the resulting echo signals using 64 multiplexed receive elements. The ASIC digitizes the received signals locally, providing more robust communication than prior analog approaches. Measurements show that the ASIC effectively switches transmit pulses up to 30 V, and digitizes echo signals with a bandwidth of 16 MHz, while consuming only 10 mW. Acoustic measurements in combination with a prototype transducer array demonstrate pulse transmission and reception. Finally, a B-mode image of a needle phantom demonstrates the imaging capability.

A single-cable PVDF transducer readout IC for intravascular photoacoustic imaging

This paper presents a custom-designed single-cable readout IC for the reception of the broadband photoacoustic (PA) signal in intravascular photoacoustic (IVPA) imaging. The readout IC is intended for direct integration behind a broadband polyvinylidene fluoride (PVDF) transducer in an IVPA catheter tip to match the impedance between the small PVDF element and the connecting cable. The capability of the readout IC to work with a single cable that combines the output signal and the power supply ensures the mechanical flexibility of the IVPA catheter. Electrical measurements show that the readout IC provides a flat frequency response from 1 MHz to 20 MHz with a 6 mA external current supply. The acoustical measurements involving the readout IC and the PVDF transducer demonstrate a 60 dB dynamic range, a sensitivity of 3.8 μV/Pa at 2.25 MHz, and a broad receiving bandwidth from 2 MHz to 15 MHz.