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Blood Contrast Enhancement with a Novel, Non-Gaseous Nanoparticle Contrast Agent
Blood Contrast Enhancement with a Novel, Non-Gaseous Nanoparticle Contrast Agent1 Samuel A. Wickline, MD, Michael Hughes, PhD, Francis C. Ngo, MD, Christopher S. Hall, PhD Jon N. Marsh, PhD, Peggy A. Brown, John S. Allen, BS, Mark D. McLean, Michael J. Scott, BS Ralph W. Fuhrhop, Gregory M. Lanza, MD, PhD
RATIONALE AND OBJECTIVES Modern ultrasound contrast agents primarily comprise microbubble formulations that circulate in the intravascular compartment and are designed to enhance acoustic signals reflected from the blood pool. A variety of shell materials have been utilized to stabilize gas bubbles of the order of 1–10 microns in diameter. Reflectivity from microbubbles is enhanced by resonance and non-linear physical effects. However, the overall efficacy of bubbles as contrast agents must be considered in light of their marked instability to insonification pressures, marked attenuation artifacts, “blooming” effects, and their short circulatory half-life. Low molecular weight gaseous perfluorocarbon formulations have been utilized in vivo because they may offer advantages in formulation and reflectivity. In contrast, higher molecular weight perfluorocarbon emulsions that are liquid at body temperature have been formulated as nongaseous nanoparticle preparations (diameters ⫽ 100 – 300 nanometers), originally for use as blood substitutes. Unfortunately they exhibit low inherent echogenicity and are poor blood pool contrast agents under conditions of conventional 2-D echocardiography or harmonic imaging, or when imaged with color flow or spectral Doppler. Nevertheless, these nanoparticle formulations are chemically inert, manifest long circulatory half-lives, are not destroyed by ultrasonic imaging, and they possess low acoustic attenuation. Such features might still render them Acad Radiol 2002; 9(suppl 2):S290 –S293 1 From the Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, MO 63146. Address correspondence to S.A.W.
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of interest as blood pool contrast agents if properly formulated and imaged. Recently, a new ultrasonic imaging modality, Power Doppler Harmonic Imaging (PDHI), has been introduced (4). This technique color-encodes changes in acoustic signal amplitude and motion of ultrasonic scatterers between insonifying pulses. PDHI has been used in a number of clinical studies to assess coronary artery bypass graft patency, tumor blood flow, and myocardial perfusion. In view of the exquisite sensitivity of Doppler for detecting the presence of small scatterers with limited scattering cross-sections as compared to microbubbles (e.g., red blood cells), and the enhanced ability of PDHI to register backscatter power, we hypothesized that certain liquid perfluorocarbon nanoparticle emulsions (5) might be more efficiently detected with this new imaging modality. Furthermore, although we have demonstrated previously that the liquid nanoparticle emulsions do not manifest any appreciable resonance behavior at clinically relevant imaging frequencies, they have performed well as targeted imaging agents in vitro and in vivo over a very broad range of frequencies (5–50 MHz)(6 – 8). Thus we anticipated that the PDHI method might permit imaging of these nanoparticles in the blood pool without reliance on any intrinsic resonance behavior.
MATERIALS AND METHODS Two versions of nanoparticles were created: a larger and a smaller variety. The perfluorocarbon nanoparticle emulsion comprised perfluorooctylbromide (40% w/v, PFOB, 3M) and a surfactant co-mixture (2.0%, w/v) and glycerin (1.7%, w/v). The surfactant co-mixture included 65 mole% lecithin (Pharmacia Inc), 35 mole% cholesterol
NANOPARTICLE CONTRAST AGENT
Academic Radiology, Vol 9, Suppl 2, 2002
Figure 1. Particle size distributions of larger (⬃450 nm) and smaller (⬃250 nm) nanoparticles.
(Sigma Chemical Co.), which was dissolved in chloroform. The chloroform-lipid mixture was evaporated, dried and dispersed into 50 mM phosphate buffer pH 7.0 (larger nanoparticles) or deionized water (smaller nanoparticles) by sonication. The suspension was transferred into a blender cup (Dynamics Corporation of America) with perfluorooctylbromide and 50 mM phosphate buffer or deionized water (as above), pre-emulsified for 30 to 60 seconds, and then transferred to an S100 Microfluidics emulsifier (Microfluidics Co.) and continuously processed at 20,000 PSI for three minutes. The completed emulsions were vialed, blanketed with nitrogen, and sealed with stopper crimp seal until use. Particle sizes were determined in triplicate at 37°C with a laser light scattering submicron particle size analyzer (Malvern Zetasizer 4, Malvern Instruments Ltd, Southborough, MA), which indicated a highly reproducible size distribution with average diameters around 465 nm (Fig 1, left) or 232 nm (Fig 1, right), respectively. The concept of using perfluorocarbon nanoparticles as a blood pool contrast agent using harmonic power Doppler imaging was piloted in vitro. The experimental set-up consisted of a 60 cc syringe positioned in a syringe pump (Model 22, Harvard Instruments, S. Natick, MA) and connected through small bore plastic tubing to a 22g plastic catheter (0.8 mm i.d.). The output orifice of the catheter was positioned immediately below the surface of a degassed, vertical water bath (2.5 cm diameter by 6 cm length). A sheer latex film was applied to the lower as-
pect of the water bath. Ultrasonic imaging through the latex was directed perpendicular to the catheter and focused at its orifice. Imaging was performed with an Acuson Sequoia and 5 MHz (5V2c) phased-array transducer operating in harmonic mode (2.5/5.0 MHz). PDHI of the 40% perfluorocarbon emulsions were evaluated and compared with a degassed saline control. In vivo experiments were performed in 10 dogs weighing 20 –25 kg anesthetized with sodium pentobarbital (30 mg/kg, i.v.) and 1% halothane in oxygen. Animals received intravenous 10 cc injections (0.5 ml/kg) of the smaller (n ⫽ 7) or larger (n ⫽ 3) perfluorocarbon nanoparticles. Ultrasonic imaging was performed with an Acuson Sequoia and 5 MHz (5V2c) phased-array transducer operating in Power Doppler Harmonic Image mode. All animals were imaged at baseline and 15, 30 and 60 minutes after the perfluorocarbon nanoparticle treatment. 2-D echocardiographic images were produced with fixed transmit, receive and time-gain compensation levels and recorded onto Super-VHS videotape and digital optical magnetic media for playback and image analysis. RESULTS Figure 2 (left) depicts the water bath into which degassed saline (control) was infused at a rate of 26 ml/min. PDHI of saline injected into the water bath was undetectable. Infusion of perfluorocarbon nanoparticles (⬃450 nm) into the water bath and imaging with PDH revealed a
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Figure 2. PDHI (2.5/5.0 MHz) of saline (left) (PDHI) or ⬃450 nm perfluorocarbon nanoparticles injected into a water phantom.
Figure 3. Left: Apical four chamber view (PDHI). Right: Opacification after administration of the ⬃450 nm nanoparticles (PDHI).
bright plume of contrast (Fig 2, right panel). The smaller nanoparticles were somewhat less detectable in vitro (in saline) but could be visualized as well. Additional experiments revealed that other imaging modalities (fundamental, harmonic, Doppler) failed to reveal any contrast effect in vitro with similar injection regimens and settings. In contrast, Optison injections resulted in visualization with all imaging modalities as expected. The efficacy of perfluorocarbon nanoparticles at two particle sizes (⬃250 nm vs ⬃450 nm) was evaluated using a canine model with Power Doppler Harmonic Imaging (2.5/5.0 MHz). Prior to contrast, the left ventricular
S292
cavity was suboptimally detected by fundamental, harmonic and Power Doppler Harmonic Imaging. Intravenous administration of the perfluorocarbon nanoparticles (⬃450 nm) produced a clear enhancement and delineation of the left ventricular cavity and endocardial borders (Fig 3). This contrast effect persisted unchanged throughout the imaging period (one hour). The contrast effect was unaffected by continuous insonification at maximum power output settings. Seven dogs treated with the ⬃250 nm nanoparticles had minimal or no incremental LV opacification. Selected animals receiving twice the dosage of the smaller particles, 20 ml (1.0 ml/kg), demonstrated
NANOPARTICLE CONTRAST AGENT
Academic Radiology, Vol 9, Suppl 2, 2002
no change in the blood pool contrast effect. These findings indicate that larger nanoparticles with a mean particle size of around 400 nm may be efficacious as blood pool contrast agents when used with Power Doppler Harmonic Imaging. CONCLUSION Existing microbubble contrast agents offer outstanding acoustic contrast in vivo, but they have short circulation persistence and are destroyed by ultrasound imaging. Mattrey and his colleagues previously demonstrated that liquid perfluorocarbon particles exhibited low inherent acoustic reflectivity when used with conventional 2-D fundamental imaging and possessed little benefit for enhancing spectral Doppler signals (1–3). Only at concentrations of 3.1% blood volume (2.0 ml/kg) did PFOB particles provide adequate ultrasonic contrast. In the present study, we have shown that liquid perfluorocarbon nanoparticles can provide excellent blood pool contrast when imaged with Harmonic Power Doppler at dosages of 0.5 ml/kg (approximately 0.7% blood volume). The contrast effect is unaffected by continuous ultrasonic imaging at high transducer power outputs and persists for at least one hour. The unique acoustic contrast effect may require nanoparticles with mean diameters
around 400 nm. Given their long circulatory persistence and acoustic stability, liquid perfluorocarbon nanoparticles may offer an alternative class of ultrasonic contrast agents for blood pool imaging and perfusion that merit continued investigation. REFERENCES 1. Mattrey R, Scheible F, Gosink B, Leopold G, Long D, Higgins C. Perfluoroctylbromide: A liver/spleen-specific and tumor-imaging ultrasound contrast material. Radiology 1982; 145:759 –762. 2. Andre M, Nelson T, Mattrey R. Physical and acoustical properties of perfluoroctylbromide, an ultrasound contrast agent. Invest Radiol 1990; 25:983–987. 3. Andre M, Steinbach G, Mattrey R. Enhancement of the echogenicity of flowing blood by the contrast agent perflubron. Invest Radiol 1993; 28: 502–506. 4. Mor-Avi V, Lang R. Recent advances in echocardiographic evaluation of left ventricular anatomy, perfusion, and function. Cardiology In Review, 2001; 9:146 –159. 5. Lanza GM, Wallace KD, Scott MJ, et al. A novel site-targeted ultrasonic contrast agent with broad biomedical application. Circulation 1996; 95: 3334 –3340. 6. Lanza GM, Wallace KD, Fischer SE, et al. High frequency ultrasonic detection of thrombi with a targeted contrast system. Ultrasound Med Biol 1997; 23:863– 870. 7. Lanza GM, Abendschein DR, Hall CH, et al. Molecular imaging of stretch-induced tissue factor expression in carotid arteries with intravascular ultrasound. Invest Radiol 2000; 35:227–234. 8. Lanza GM, Abendschein DR, Hall CH, 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 – 614.
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RATIONALE AND OBJECTIVES Modern ultrasound contrast agents primarily comprise microbubble formulations that circulate in the intravascular compartment and are designed to enhance acoustic signals reflected from the blood pool. A variety of shell materials have been utilized to stabilize gas bubbles of the order of 1–10 microns in diameter. Reflectivity from microbubbles is enhanced by resonance and non-linear physical effects. However, the overall efficacy of bubbles as contrast agents must be considered in light of their marked instability to insonification pressures, marked attenuation artifacts, “blooming” effects, and their short circulatory half-life. Low molecular weight gaseous perfluorocarbon formulations have been utilized in vivo because they may offer advantages in formulation and reflectivity. In contrast, higher molecular weight perfluorocarbon emulsions that are liquid at body temperature have been formulated as nongaseous nanoparticle preparations (diameters ⫽ 100 – 300 nanometers), originally for use as blood substitutes. Unfortunately they exhibit low inherent echogenicity and are poor blood pool contrast agents under conditions of conventional 2-D echocardiography or harmonic imaging, or when imaged with color flow or spectral Doppler. Nevertheless, these nanoparticle formulations are chemically inert, manifest long circulatory half-lives, are not destroyed by ultrasonic imaging, and they possess low acoustic attenuation. Such features might still render them Acad Radiol 2002; 9(suppl 2):S290 –S293 1 From the Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, MO 63146. Address correspondence to S.A.W.
©
AUR, 2002
S290
of interest as blood pool contrast agents if properly formulated and imaged. Recently, a new ultrasonic imaging modality, Power Doppler Harmonic Imaging (PDHI), has been introduced (4). This technique color-encodes changes in acoustic signal amplitude and motion of ultrasonic scatterers between insonifying pulses. PDHI has been used in a number of clinical studies to assess coronary artery bypass graft patency, tumor blood flow, and myocardial perfusion. In view of the exquisite sensitivity of Doppler for detecting the presence of small scatterers with limited scattering cross-sections as compared to microbubbles (e.g., red blood cells), and the enhanced ability of PDHI to register backscatter power, we hypothesized that certain liquid perfluorocarbon nanoparticle emulsions (5) might be more efficiently detected with this new imaging modality. Furthermore, although we have demonstrated previously that the liquid nanoparticle emulsions do not manifest any appreciable resonance behavior at clinically relevant imaging frequencies, they have performed well as targeted imaging agents in vitro and in vivo over a very broad range of frequencies (5–50 MHz)(6 – 8). Thus we anticipated that the PDHI method might permit imaging of these nanoparticles in the blood pool without reliance on any intrinsic resonance behavior.
MATERIALS AND METHODS Two versions of nanoparticles were created: a larger and a smaller variety. The perfluorocarbon nanoparticle emulsion comprised perfluorooctylbromide (40% w/v, PFOB, 3M) and a surfactant co-mixture (2.0%, w/v) and glycerin (1.7%, w/v). The surfactant co-mixture included 65 mole% lecithin (Pharmacia Inc), 35 mole% cholesterol
NANOPARTICLE CONTRAST AGENT
Academic Radiology, Vol 9, Suppl 2, 2002
Figure 1. Particle size distributions of larger (⬃450 nm) and smaller (⬃250 nm) nanoparticles.
(Sigma Chemical Co.), which was dissolved in chloroform. The chloroform-lipid mixture was evaporated, dried and dispersed into 50 mM phosphate buffer pH 7.0 (larger nanoparticles) or deionized water (smaller nanoparticles) by sonication. The suspension was transferred into a blender cup (Dynamics Corporation of America) with perfluorooctylbromide and 50 mM phosphate buffer or deionized water (as above), pre-emulsified for 30 to 60 seconds, and then transferred to an S100 Microfluidics emulsifier (Microfluidics Co.) and continuously processed at 20,000 PSI for three minutes. The completed emulsions were vialed, blanketed with nitrogen, and sealed with stopper crimp seal until use. Particle sizes were determined in triplicate at 37°C with a laser light scattering submicron particle size analyzer (Malvern Zetasizer 4, Malvern Instruments Ltd, Southborough, MA), which indicated a highly reproducible size distribution with average diameters around 465 nm (Fig 1, left) or 232 nm (Fig 1, right), respectively. The concept of using perfluorocarbon nanoparticles as a blood pool contrast agent using harmonic power Doppler imaging was piloted in vitro. The experimental set-up consisted of a 60 cc syringe positioned in a syringe pump (Model 22, Harvard Instruments, S. Natick, MA) and connected through small bore plastic tubing to a 22g plastic catheter (0.8 mm i.d.). The output orifice of the catheter was positioned immediately below the surface of a degassed, vertical water bath (2.5 cm diameter by 6 cm length). A sheer latex film was applied to the lower as-
pect of the water bath. Ultrasonic imaging through the latex was directed perpendicular to the catheter and focused at its orifice. Imaging was performed with an Acuson Sequoia and 5 MHz (5V2c) phased-array transducer operating in harmonic mode (2.5/5.0 MHz). PDHI of the 40% perfluorocarbon emulsions were evaluated and compared with a degassed saline control. In vivo experiments were performed in 10 dogs weighing 20 –25 kg anesthetized with sodium pentobarbital (30 mg/kg, i.v.) and 1% halothane in oxygen. Animals received intravenous 10 cc injections (0.5 ml/kg) of the smaller (n ⫽ 7) or larger (n ⫽ 3) perfluorocarbon nanoparticles. Ultrasonic imaging was performed with an Acuson Sequoia and 5 MHz (5V2c) phased-array transducer operating in Power Doppler Harmonic Image mode. All animals were imaged at baseline and 15, 30 and 60 minutes after the perfluorocarbon nanoparticle treatment. 2-D echocardiographic images were produced with fixed transmit, receive and time-gain compensation levels and recorded onto Super-VHS videotape and digital optical magnetic media for playback and image analysis. RESULTS Figure 2 (left) depicts the water bath into which degassed saline (control) was infused at a rate of 26 ml/min. PDHI of saline injected into the water bath was undetectable. Infusion of perfluorocarbon nanoparticles (⬃450 nm) into the water bath and imaging with PDH revealed a
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Academic Radiology, Vol 9, Suppl 2, 2002
Figure 2. PDHI (2.5/5.0 MHz) of saline (left) (PDHI) or ⬃450 nm perfluorocarbon nanoparticles injected into a water phantom.
Figure 3. Left: Apical four chamber view (PDHI). Right: Opacification after administration of the ⬃450 nm nanoparticles (PDHI).
bright plume of contrast (Fig 2, right panel). The smaller nanoparticles were somewhat less detectable in vitro (in saline) but could be visualized as well. Additional experiments revealed that other imaging modalities (fundamental, harmonic, Doppler) failed to reveal any contrast effect in vitro with similar injection regimens and settings. In contrast, Optison injections resulted in visualization with all imaging modalities as expected. The efficacy of perfluorocarbon nanoparticles at two particle sizes (⬃250 nm vs ⬃450 nm) was evaluated using a canine model with Power Doppler Harmonic Imaging (2.5/5.0 MHz). Prior to contrast, the left ventricular
S292
cavity was suboptimally detected by fundamental, harmonic and Power Doppler Harmonic Imaging. Intravenous administration of the perfluorocarbon nanoparticles (⬃450 nm) produced a clear enhancement and delineation of the left ventricular cavity and endocardial borders (Fig 3). This contrast effect persisted unchanged throughout the imaging period (one hour). The contrast effect was unaffected by continuous insonification at maximum power output settings. Seven dogs treated with the ⬃250 nm nanoparticles had minimal or no incremental LV opacification. Selected animals receiving twice the dosage of the smaller particles, 20 ml (1.0 ml/kg), demonstrated
NANOPARTICLE CONTRAST AGENT
Academic Radiology, Vol 9, Suppl 2, 2002
no change in the blood pool contrast effect. These findings indicate that larger nanoparticles with a mean particle size of around 400 nm may be efficacious as blood pool contrast agents when used with Power Doppler Harmonic Imaging. CONCLUSION Existing microbubble contrast agents offer outstanding acoustic contrast in vivo, but they have short circulation persistence and are destroyed by ultrasound imaging. Mattrey and his colleagues previously demonstrated that liquid perfluorocarbon particles exhibited low inherent acoustic reflectivity when used with conventional 2-D fundamental imaging and possessed little benefit for enhancing spectral Doppler signals (1–3). Only at concentrations of 3.1% blood volume (2.0 ml/kg) did PFOB particles provide adequate ultrasonic contrast. In the present study, we have shown that liquid perfluorocarbon nanoparticles can provide excellent blood pool contrast when imaged with Harmonic Power Doppler at dosages of 0.5 ml/kg (approximately 0.7% blood volume). The contrast effect is unaffected by continuous ultrasonic imaging at high transducer power outputs and persists for at least one hour. The unique acoustic contrast effect may require nanoparticles with mean diameters
around 400 nm. Given their long circulatory persistence and acoustic stability, liquid perfluorocarbon nanoparticles may offer an alternative class of ultrasonic contrast agents for blood pool imaging and perfusion that merit continued investigation. REFERENCES 1. Mattrey R, Scheible F, Gosink B, Leopold G, Long D, Higgins C. Perfluoroctylbromide: A liver/spleen-specific and tumor-imaging ultrasound contrast material. Radiology 1982; 145:759 –762. 2. Andre M, Nelson T, Mattrey R. Physical and acoustical properties of perfluoroctylbromide, an ultrasound contrast agent. Invest Radiol 1990; 25:983–987. 3. Andre M, Steinbach G, Mattrey R. Enhancement of the echogenicity of flowing blood by the contrast agent perflubron. Invest Radiol 1993; 28: 502–506. 4. Mor-Avi V, Lang R. Recent advances in echocardiographic evaluation of left ventricular anatomy, perfusion, and function. Cardiology In Review, 2001; 9:146 –159. 5. Lanza GM, Wallace KD, Scott MJ, et al. A novel site-targeted ultrasonic contrast agent with broad biomedical application. Circulation 1996; 95: 3334 –3340. 6. Lanza GM, Wallace KD, Fischer SE, et al. High frequency ultrasonic detection of thrombi with a targeted contrast system. Ultrasound Med Biol 1997; 23:863– 870. 7. Lanza GM, Abendschein DR, Hall CH, et al. Molecular imaging of stretch-induced tissue factor expression in carotid arteries with intravascular ultrasound. Invest Radiol 2000; 35:227–234. 8. Lanza GM, Abendschein DR, Hall CH, 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 – 614.
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