Polymer Microbubbles as Dual Modal Contrast Agent for Ultrasound and Computed Tomography

• The hybrid imaging combines the anatomical information with the functional or metabolic information using different conventional single imaging modalities improving the overall diagnosis outcome of the clinical examination. Since the introduction of the first hybrid imaging device PET-CT in 1998 different combinations of hybrid imaging were developed such as PET-MRI, SPECT-CT.However, lack of multimodal contrast agent specifically aimed for hybrid imaging limits the diagnostic outcome of these novel techniques. Initial attempts in fabrication of hybrid contrast agents were made by combining previously existing single modal contrast agents into one. In this study, polyvinyl alcohol (PVA) microbubbles (MB) and gold nanoparticles - which by themselves are already established contrast agents used in preclinical studies for ultrasound and CT, respectively - were chosen as parent contrast agents to fabricate the dual modal Contrast Agent for UltraSound and CT (CACTUS).MethodThe fabrication of MBs was adapted from Cavalieri et al.[1]. PVA powder (Sigma Aldrich, MO USA) was dissolved in the water at 80°C. The aqueous PVA-chains were cleaved by sodium metaperiodate (NaIO4, purity>99.0%, Sigma Aldrich, MO USA). Vigorous stirring force was applied to the resulting telechelic aldehydic PVA-chains for 2 hours to crosslink the telechelic aldehydic PVA-chains and form the PVA-coated MBs at the water-air interface. CACTUS MBs were synthesized in a similar fashion to the above, but adding gold nanoparticles (diameter 1.9nm, Nanoprobes, NY, USA) during formation of the MBs.The size distributions of MBs and CACATUS MBs were determined using an optical microscope (ECLIPSE Ci-S, Nikon, Tokyo, Japan) and a Neubauer counting chamber (Brand GmbH, Wertheim, Germany).The acoustic attenuation coefficients of the MBs suspension were acquired at peak negative pressure (PNP) from 10 - 300 kPa. Three MBs suspension samples with concentrations of (sample A),  (sample B) and  ml-1 (sample C) were prepared and loaded in a 1 cm thick two-cavity chamber. A flat single crystal ultrasound transducer with central frequency 3.5MHz was used to generate the ultrasound beam. The amplitude of received echoes through samples and water were compared at the fundamental frequency, as well as the 2nd and 3rd harmonic for each value of the concentration used.The mass attenuation of water, suspension of gold nanoparticles with concentration 160mg/L, plain MBs, and CACTUS MBs, was measured by quantum FX-CT micro-CT (PerkinElmer Inc, MA, USA). The micro-CT was operated at a current of 200mA with exposure time of 120s and varied voltage 50kV, 70kV and 90kV. Each 3D image has a size of 512*512*512 pixels or 75.8*75.8*75.8 mm. Contrast to noise ratios (CNR) between water and all samples were calculated following Eq. 1.Where S(x,y,z) and W(x,y,z) are the mass attenuation of the sample and water per voxel, respectively. ns(x,y,z) and nw(x,y,z) are the noise function with zero mean of sample and water respectively. Ms and Mw are the mean mass attenuation acquired for the sample and water in the volume of interest. The σs2 and σw2 are the variance of the mass attenuation read out of the sample and water in the volume of interested.In addition to the gas-core MBs for the CT tests, liquid-core gold loaded capsules were synthesized in two steps. In the first step, PVA shelled liquid-core capsules were obtained by exposing MBs to 66% v/v ethanol solution. In the second step, the resulting liquid-core capsules were mixed with high concentration gold nanoparticles suspension and homogenized by a shaker (MS 3 basic, IKA, Königswinter Germany) at 500rpm for 1 hour for goal loading. The resulting gold loaded capsules were washed with Milli-Q water using centrifuge (Galaxy 5D digital microcentrifuge, VWR, USA) at a speed of 1000 g for 5 min.Results and discussionThe mean diameter of MBs is 3.6±1.1 μm. The mean diameter of CACTUS MBs is 3.2±0.7 μm. The size distribution of the gold loaded capsules was not investigated separately, but rather assumed identical to the plain MBs. The number and the volume distribution of MBs and CACTUS MBs are shown in figure 1. The results demonstrate that most of the CACTUS MBs and MBs have a diameter from 1 to 6 μm. Therefore, they are able pass through the capillaries and will resonate within typical clinical diagnostic ultrasound frequency below 15 MHz.Pressure dependent acoustic attenuation coefficients of the sample A, B, and C are shown in figure 2. The results show that attenuation coefficients of sample A and B at the fundamental frequency stay constant and slightly increase at the second harmonic at the PNP below 100kPa, indicating a linear oscillation of MBs. As the PNP reaches 200kPa, the attenuation coefficient of sample A at fundamental frequency decreases while at 2nd and 3rd harmonics increases, indicating that the energy of the echo shifts from the fundamental frequency to the 2nd and 3rd harmonics. As the PNP goes higher to 300kPa, the attenuation coefficient of sample A at the fundamental frequency, 2nd, and 3rd harmonics decreases, suggesting that the energy shifts to an even higher harmonic. At the same time, the attenuation coefficient of sample B stays constant at fundamental frequency, decreases at 2nd harmonics, and increases at the 3rd harmonic, suggesting the energy starts to shift to the 3rd harmonic. The attenuation coefficient of sample C at fundamental frequency, 2nd and 3rd harmonics keep constant and low due to low sample concentration. The test reveals the energy shifting of the echo to the higher harmonics at PNP higher than 100 kPa, indicating the nonlinear oscillation of MBs at PNP higher than 100 kPa. Moreover, the concentration of the MBs seems to influence the energy shifting: the higher the concentration the earlier the shift to the higher harmonics occurs, in the range of the concentration consider in this study.The pilot results of the micro-CT tests are presented in Table 1. The reference, gold nanoparticles solution, has the highest CNR per voxel at all CT operating voltages. The CNR per voxel of CACTUS MBs suspensions is below 0.1, virtually equaling the MBs at all operating voltages, suggesting that no gold or very little gold were loaded into the shell of the CACTUS MBs. The gold loaded capsules suspension has higher CNR per voxel than the capsule supernatant (the surrounding environment of capsules) and the MBs suspension, implying that the gold nanoparticles were loaded into the capsules. However, it is not clear whether the gold nanoparticles were loaded in the core of the MBs or in the MBs shell. The expected sharp increase of CNR per voxel at the k-edge of gold did not appear. We believe that is because even at our highest operating voltage of 90kV, the percentage of the photons with energy higher than 80.7 keV is still low. Introduction of a high-pass metal filter could increase the percentage of high energy photon. On the other hand, the metal filter will reduce the total number of the photons which would increase the noise of the images. Since same current was applied on every CT test, less X-ray photons reached the sensors when the CT was operated at low voltage. Therefore, it might be worth performing additional calibration tests to adjust the operating currents to make sure that the numbers of the photons that reach the sensor at every operating voltage are the same.ConclusionIn this study, the CACTUS MBs and gold loaded capsules were fabricated as potential candidates for dual modal contrast agent. The characterization revealed that gold loaded capsule is a promising initial step. Nevertheless, the method to convert back liquid-core capsules to gas-core MBs needs to be established.[1] Cavalieri, F., El Hamassi, A., Chiessi, E., Paradossi, G., Villa, R., & Zaffaroni, N. (2006). Tethering functional ligands onto shell of ultrasound active polymeric microbubbles. Biomacromolecules, 7(2), 604-611.

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TEKNIK OCH TEKNOLOGIER  -- Medicinteknik -- Annan medicinteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Medical Engineering -- Other Medical Engineering (hsv//eng)

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