HHS Public Access Author manuscript Author Manuscript
Circ Cardiovasc Imaging. Author manuscript; available in PMC 2017 January 01. Published in final edited form as: Circ Cardiovasc Imaging. 2016 January ; 9(1): . doi:10.1161/CIRCIMAGING.115.004374.
PHASE-CONVERSION NANOPARTICLE CONTRAST AGENTS: DO GOOD THINGS COME IN SMALL PACKAGES? Jonathan R. Lindner, MD Knight Cardiovascular Institute, Oregon Health & Science University; Portland, OR
Author Manuscript
Ultrasound contrast agents composed of encapsulated microbubbles are now widely used in many parts of the world for an array of clinical applications. During diagnostic echocardiography, contrast agents are used to better delineate endocardial borders and intracavitary masses. Echocardiographic laboratories that have specialized skill and knowledge can also apply commercially-produced microbubble contrast agents in an offlabel fashion for myocardial perfusion imaging in order to detect rest or stress-induced ischemia in coronary artery disease, microvascular reflow, tissue viability, tumor perfusion, etc.
Author Manuscript
The ultrasound contrast agents that have been used in humans vary in terms of their composition, including the content of their gas core (perfluorocarbons, SF6) although all are high-molecular weight gases with relatively low solubility and diffusivity in order to improve stability in vivo.1 There are also differences in encapsulation strategy (albumin, lipid), although a common thread has been that the viscoelastic properties must be appropriate to allow non-linear oscillation even during low mechanical index (MI) imaging.1 Commercially produced agents also vary in terms of size and size dispersion. It is important that the vast majority of microbubbles be smaller than the dimensions of the microvessels through which they travel and yet very small (sub-micron) bubbles are less stable due to surface tension and contribute relatively little to signal enhancement.
Author Manuscript
In this issue of Circulation: Cardiovascular Imaging, Porter et al.2 report results from a collaborative study where signal enhancement during myocardial contrast echocardiography (MCE) was examined with a novel phase-shifting nanodroplet. This class of agents differ from microbubbles in that they are almost an order of magnitude smaller in diameter, have a predominately liquid-phase core, and require activation whereby diameter increases upon conversion of the core to gas phase (vaporization) during the pressure oscillations of the acoustic field.3 By virtue of their size, these agents can potentially have extravascular access which may allow unique opportunities for contrast ultrasound imaging or for ultrasoundfacilitated gene or drug delivery for disease states (cancer, inflammatory diseases) or tissues (liver, renal glomeruli) where microvascular permeability is enhanced. One could also imagine that site-targeted acoustic delivery of drug or gene could be further enhanced by
Correspondence to: Jonathan R. Lindner, MD, Knight Cardiovascular Institute, UHN 62, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, P – (503) 494-8750, F – (503) 494-8550, [email protected]. DISCLOSURES None.
Lindner
Page 2
Author Manuscript
phase conversion of nano-droplets directly upstream from the tissue of interest, particularly if the post-activated agent was sufficiently large to allow entrapment.
Author Manuscript
The study by Porter et. al.2 is the first to examine acoustic activation of nanodroplets within the ventricular cavity and subsequent myocardial enhancement in a porcine model. There are several important findings of this study. Although they did not perform a systematic evaluation of optimal conditions, the study is the first to show that conventional imaging parameters that are used in patients can activate the agent in the left ventricular cavity and subsequently image it in the myocardial microcirculation. The authors have also provided important preliminary in vivo information on how ultrasound activation of the nanodroplets is dependent on acoustic pressure. On a practical note, the agent used in the study by Porter et al. is relatively easy to prepare based on low-temperature condensation of gases that are already used in commercially-produced agents. Finally, at least one of the agents that was tested was relatively easy to vaporize with ultrasound in the diagnostic range due to the relatively low boiling point of the octafluoropropane (C3F8) when compared to similar compounds that have been used such as dodecafluoropentane (C5F12) which is generally liquid at room temperature or to the condensed decafluorobutane (C4F10) agent which Porter and colleagues also tested.
Author Manuscript
An important contextual issue raised by the study by Porter and colleagues is the question of “why?”. Is there much to be gained by using nanodroplets that have the added complexity of needing to be activated in vivo? In their justification, the authors are probably correct (at least for polydisperse contrast agents) that a large proportion of the agent is too labile to contribute to signal generation during the real-time low-power imaging methods that are now routinely used for MCE perfusion imaging. However, this is probably not a major drawback for commercially-produced microbubble agents that have a much narrower size distribution, nor is there anything necessarily wrong with simply compensating with higher dose. Other theoretical potential advantages of nanodroplets must be considered. Based on the knowledge of size-dependent clearance of liposomes, one would predict that nanoscale agents would circulate longer than conventional 1-5 μm diameter microbubbles.4 This feature could potentially improve the stability of the blood pool concentration to the point of achieving stable concentration after a single bolus injection. Unfortunately, the loss of the ultrasound signal from nanodroplets in under three minutes in this study indicated that reticuloendothelial clearance was still rapid.
Author Manuscript
A significant limitation in the current practice of MCE perfusion imaging is that high microbubble concentration within the left or right ventricle produces attenuation of far-field territories. The use of nanodroplet phase-conversion agents could be an advantage. Cavity attenuation could be minimized if the contrast agent is activated at high MI and then imaged in the myocardial microcirculation seconds later at a lower MI. Also, it is known that the relationship between microbubble signal generation (scattering cross-section) and attenuation (absorption and extinction cross-section) are variably related based size. The ability to generate ideally-sized microbubbles that need to be stable for only the brief time of transfer from LV to myocardium may reduce far-field shadowing.5,6 Amelioration of attenuation is likely to be a topic addressed in the next phase of investigation, although some
Circ Cardiovasc Imaging. Author manuscript; available in PMC 2017 January 01.
Lindner
Page 3
Author Manuscript
of the illustrations of MCE imaging in the Porter study hint at relatively low posterior wall shadowing. As one would expect with such a revolutionary concept, the application of nanodroplets during MCE perfusion imaging begs many questions. The optimal acoustic conditions for activation including frequency, acoustic pressure, pulse duration are unclear; as are the effects of multipulse versus single-pulse imaging (each of which were used in this study in the form of power modulation and harmonic imaging). Since the dose was quantified simply as a volume (20% solution), it is not possible to compare signal to that from microbubbles in equivalent dose (either based on particle number or perfluorocarbon mass).
Author Manuscript Author Manuscript
One critical issue that remains unresolved is the size of the microbubbles that are formed during ventricular activation. Previous studies with these perfluorocarbon nanodroplets have found that acoustic pressure influences not only the amount but also the size of vaporized microbubbles in an inverse manner.7 Hence, one could surmise that microbubble size will vary considerably between patients based on body morphometrics that influence damping of ultrasound and that are not replicated in an animal study. The safety concern raised by unpredictable microbubble diameter (which can range between 1 and 20 μm in in vitro studies)7 is not entirely solved in this study. The similar rates of decline in intensity from blood pool and myocardium illustrated in Figure 5 by Porter and colleagues was used to argue that microbubbles were freely-circulating. An alternative explanation is that microbubbles that were entrapped based on size were being continuously destroyed by the repetitive high-MI imaging pulses that were used to vaporize and image the bubbles. Only by shifting the ultrasound beam to an unexposed region and not seeing an abrupt increase in signal or by a delayed pause could one exclude microbubble lodging which can occur with bubbles as small as 5 μm.8
Author Manuscript
The investigators of this study have adeptly noted that standard quantitative perfusion imaging with MCE cannot be performed with nanodroplets. If a single long activation sequence is performed followed by low MI imaging, then the microbubble concentration of blood pool entering the myocardium will vary too much over the time needed for standard destruction-replenishment approaches. One can use high-MI intermittent imaging (triggered to ECG) to simultaneously activate agent and image the microcirculation. However, changes in blood pool concentration of vaporized bubbles will again be expected caused by the progressive prolongation of the pulsing interval. There is the possibility of activating nanodroplets in the cavity and then measuring the transfer kinetics through the myocardium using low MI imaging. Unfortunately, this mathematical model measures flow per unit volume rather than flow itself unless one has the means to separately measure compartment volume and input function.9 An interesting finding of the study was that robust signal enhancement was achieved with continuous high-MI imaging which is not possible with conventional microbubbles. Continuous imaging at MI 1.1 should destroy microbubbles, if not in the cavity then during their slow microvascular transit. The finding of signal enhancement with these stetting implies that vaporized compared to standard microbubbles are either more resistant to destruction, larger (and hence less destructible), more plentiful, or are formed within the
Circ Cardiovasc Imaging. Author manuscript; available in PMC 2017 January 01.
Lindner
Page 4
Author Manuscript
microcirculation itself. Unfortunately, continuous high-power imaging provides information primarily on blood volume which is likely to be useful only for detecting severe perfusion defects or infarction. However, if vaporization is occurring to a large extent within the microcirculation, then a “washout” analysis of contrast disappearance may be an alternative for introducing the kinetic information needed to assess perfusion.10 In summary, the study by Porter and colleagues provide some very intriguing and novel findings on a new approach to MCE microvascular imaging. The major questions that now must be addressed are: (1) is the formation of microbubbles reproducible and safe, and (2) does this complicated approach have any major clinical advantage to state-of-the-art techniques already in practice today?
REFERENCES Author Manuscript Author Manuscript
1. Kaufmann BA, Wei K, Lindner JR. Contrast echocardiography. Curr Probl Cardiol. 2007; 32:51–96. [PubMed: 17208647] 2. Porter TR, Arena C, Sayyed S, Lof J, High RR, Xie F, Dayton PA. Targeted transthoracic acoustic activation of systemically administered nanodroplets to detect myocardial perfusion abnormalities. 2016; 9:e003770. 3. Sheeran PS, Luois S, Dayton PA, Matsunaga TO. Formulation and acoustic studies of a new phaseshift agent for diagnostic and therapeutic ultrasound. Langmuir : the ACS journal of surfaces and colloids. 2011; 27:10412–10420. [PubMed: 21744860] 4. Longmire M, Choyke PL, Kobayashi H. Clearance properties of nano-sized particles and molecules as imaging agents: Considerations and caveats. Nanomedicine. 2008; 3:703–717. [PubMed: 18817471] 5. Sirsi S, Feshitan J, Kwan J, Homma S, Borden M. Effect of microbubble size on fundamental mode high frequency ultrasound imaging in mice. Ultrasound Med Biol. 2010; 36:935–948. [PubMed: 20447755] 6. Ainslie MA, Leighton TG. Review of scattering and extinction cross-sections, damping factors, and resonance frequencies of a spherical gas bubble. The Journal of the Acoustical Society of America. 2011; 130:3184–3208. [PubMed: 22087992] 7. Sheeran PS, Matsunaga TO, Dayton PA. Phase-transition thresholds and vaporization phenomena for ultrasound phase-change nanoemulsions assessed via high-speed optical microscopy. Physics in medicine and biology. 2013; 58:4513–4534. [PubMed: 23760161] 8. Kaufmann BA, Lankford M, Behm CZ, French BA, Klibanov AL, Xu Y, Lindner JR. Highresolution myocardial perfusion imaging in mice with high-frequency echocardiographic detection of a depot contrast agent. J Am Soc Echocardiogr. 2007; 20:136–143. [PubMed: 17275698] 9. Lindner JR, Skyba DM, Goodman NC, Jayaweera AR, Kaul S. Changes in myocardial blood volume with graded coronary stenosis. Am J Physiol. 1997; 272:H567–575. [PubMed: 9038980] 10. 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; 97:473–483. [PubMed: 9490243]
Author Manuscript Circ Cardiovasc Imaging. Author manuscript; available in PMC 2017 January 01.
Circ Cardiovasc Imaging. Author manuscript; available in PMC 2017 January 01. Published in final edited form as: Circ Cardiovasc Imaging. 2016 January ; 9(1): . doi:10.1161/CIRCIMAGING.115.004374.
PHASE-CONVERSION NANOPARTICLE CONTRAST AGENTS: DO GOOD THINGS COME IN SMALL PACKAGES? Jonathan R. Lindner, MD Knight Cardiovascular Institute, Oregon Health & Science University; Portland, OR
Author Manuscript
Ultrasound contrast agents composed of encapsulated microbubbles are now widely used in many parts of the world for an array of clinical applications. During diagnostic echocardiography, contrast agents are used to better delineate endocardial borders and intracavitary masses. Echocardiographic laboratories that have specialized skill and knowledge can also apply commercially-produced microbubble contrast agents in an offlabel fashion for myocardial perfusion imaging in order to detect rest or stress-induced ischemia in coronary artery disease, microvascular reflow, tissue viability, tumor perfusion, etc.
Author Manuscript
The ultrasound contrast agents that have been used in humans vary in terms of their composition, including the content of their gas core (perfluorocarbons, SF6) although all are high-molecular weight gases with relatively low solubility and diffusivity in order to improve stability in vivo.1 There are also differences in encapsulation strategy (albumin, lipid), although a common thread has been that the viscoelastic properties must be appropriate to allow non-linear oscillation even during low mechanical index (MI) imaging.1 Commercially produced agents also vary in terms of size and size dispersion. It is important that the vast majority of microbubbles be smaller than the dimensions of the microvessels through which they travel and yet very small (sub-micron) bubbles are less stable due to surface tension and contribute relatively little to signal enhancement.
Author Manuscript
In this issue of Circulation: Cardiovascular Imaging, Porter et al.2 report results from a collaborative study where signal enhancement during myocardial contrast echocardiography (MCE) was examined with a novel phase-shifting nanodroplet. This class of agents differ from microbubbles in that they are almost an order of magnitude smaller in diameter, have a predominately liquid-phase core, and require activation whereby diameter increases upon conversion of the core to gas phase (vaporization) during the pressure oscillations of the acoustic field.3 By virtue of their size, these agents can potentially have extravascular access which may allow unique opportunities for contrast ultrasound imaging or for ultrasoundfacilitated gene or drug delivery for disease states (cancer, inflammatory diseases) or tissues (liver, renal glomeruli) where microvascular permeability is enhanced. One could also imagine that site-targeted acoustic delivery of drug or gene could be further enhanced by
Correspondence to: Jonathan R. Lindner, MD, Knight Cardiovascular Institute, UHN 62, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, P – (503) 494-8750, F – (503) 494-8550, [email protected]. DISCLOSURES None.
Lindner
Page 2
Author Manuscript
phase conversion of nano-droplets directly upstream from the tissue of interest, particularly if the post-activated agent was sufficiently large to allow entrapment.
Author Manuscript
The study by Porter et. al.2 is the first to examine acoustic activation of nanodroplets within the ventricular cavity and subsequent myocardial enhancement in a porcine model. There are several important findings of this study. Although they did not perform a systematic evaluation of optimal conditions, the study is the first to show that conventional imaging parameters that are used in patients can activate the agent in the left ventricular cavity and subsequently image it in the myocardial microcirculation. The authors have also provided important preliminary in vivo information on how ultrasound activation of the nanodroplets is dependent on acoustic pressure. On a practical note, the agent used in the study by Porter et al. is relatively easy to prepare based on low-temperature condensation of gases that are already used in commercially-produced agents. Finally, at least one of the agents that was tested was relatively easy to vaporize with ultrasound in the diagnostic range due to the relatively low boiling point of the octafluoropropane (C3F8) when compared to similar compounds that have been used such as dodecafluoropentane (C5F12) which is generally liquid at room temperature or to the condensed decafluorobutane (C4F10) agent which Porter and colleagues also tested.
Author Manuscript
An important contextual issue raised by the study by Porter and colleagues is the question of “why?”. Is there much to be gained by using nanodroplets that have the added complexity of needing to be activated in vivo? In their justification, the authors are probably correct (at least for polydisperse contrast agents) that a large proportion of the agent is too labile to contribute to signal generation during the real-time low-power imaging methods that are now routinely used for MCE perfusion imaging. However, this is probably not a major drawback for commercially-produced microbubble agents that have a much narrower size distribution, nor is there anything necessarily wrong with simply compensating with higher dose. Other theoretical potential advantages of nanodroplets must be considered. Based on the knowledge of size-dependent clearance of liposomes, one would predict that nanoscale agents would circulate longer than conventional 1-5 μm diameter microbubbles.4 This feature could potentially improve the stability of the blood pool concentration to the point of achieving stable concentration after a single bolus injection. Unfortunately, the loss of the ultrasound signal from nanodroplets in under three minutes in this study indicated that reticuloendothelial clearance was still rapid.
Author Manuscript
A significant limitation in the current practice of MCE perfusion imaging is that high microbubble concentration within the left or right ventricle produces attenuation of far-field territories. The use of nanodroplet phase-conversion agents could be an advantage. Cavity attenuation could be minimized if the contrast agent is activated at high MI and then imaged in the myocardial microcirculation seconds later at a lower MI. Also, it is known that the relationship between microbubble signal generation (scattering cross-section) and attenuation (absorption and extinction cross-section) are variably related based size. The ability to generate ideally-sized microbubbles that need to be stable for only the brief time of transfer from LV to myocardium may reduce far-field shadowing.5,6 Amelioration of attenuation is likely to be a topic addressed in the next phase of investigation, although some
Circ Cardiovasc Imaging. Author manuscript; available in PMC 2017 January 01.
Lindner
Page 3
Author Manuscript
of the illustrations of MCE imaging in the Porter study hint at relatively low posterior wall shadowing. As one would expect with such a revolutionary concept, the application of nanodroplets during MCE perfusion imaging begs many questions. The optimal acoustic conditions for activation including frequency, acoustic pressure, pulse duration are unclear; as are the effects of multipulse versus single-pulse imaging (each of which were used in this study in the form of power modulation and harmonic imaging). Since the dose was quantified simply as a volume (20% solution), it is not possible to compare signal to that from microbubbles in equivalent dose (either based on particle number or perfluorocarbon mass).
Author Manuscript Author Manuscript
One critical issue that remains unresolved is the size of the microbubbles that are formed during ventricular activation. Previous studies with these perfluorocarbon nanodroplets have found that acoustic pressure influences not only the amount but also the size of vaporized microbubbles in an inverse manner.7 Hence, one could surmise that microbubble size will vary considerably between patients based on body morphometrics that influence damping of ultrasound and that are not replicated in an animal study. The safety concern raised by unpredictable microbubble diameter (which can range between 1 and 20 μm in in vitro studies)7 is not entirely solved in this study. The similar rates of decline in intensity from blood pool and myocardium illustrated in Figure 5 by Porter and colleagues was used to argue that microbubbles were freely-circulating. An alternative explanation is that microbubbles that were entrapped based on size were being continuously destroyed by the repetitive high-MI imaging pulses that were used to vaporize and image the bubbles. Only by shifting the ultrasound beam to an unexposed region and not seeing an abrupt increase in signal or by a delayed pause could one exclude microbubble lodging which can occur with bubbles as small as 5 μm.8
Author Manuscript
The investigators of this study have adeptly noted that standard quantitative perfusion imaging with MCE cannot be performed with nanodroplets. If a single long activation sequence is performed followed by low MI imaging, then the microbubble concentration of blood pool entering the myocardium will vary too much over the time needed for standard destruction-replenishment approaches. One can use high-MI intermittent imaging (triggered to ECG) to simultaneously activate agent and image the microcirculation. However, changes in blood pool concentration of vaporized bubbles will again be expected caused by the progressive prolongation of the pulsing interval. There is the possibility of activating nanodroplets in the cavity and then measuring the transfer kinetics through the myocardium using low MI imaging. Unfortunately, this mathematical model measures flow per unit volume rather than flow itself unless one has the means to separately measure compartment volume and input function.9 An interesting finding of the study was that robust signal enhancement was achieved with continuous high-MI imaging which is not possible with conventional microbubbles. Continuous imaging at MI 1.1 should destroy microbubbles, if not in the cavity then during their slow microvascular transit. The finding of signal enhancement with these stetting implies that vaporized compared to standard microbubbles are either more resistant to destruction, larger (and hence less destructible), more plentiful, or are formed within the
Circ Cardiovasc Imaging. Author manuscript; available in PMC 2017 January 01.
Lindner
Page 4
Author Manuscript
microcirculation itself. Unfortunately, continuous high-power imaging provides information primarily on blood volume which is likely to be useful only for detecting severe perfusion defects or infarction. However, if vaporization is occurring to a large extent within the microcirculation, then a “washout” analysis of contrast disappearance may be an alternative for introducing the kinetic information needed to assess perfusion.10 In summary, the study by Porter and colleagues provide some very intriguing and novel findings on a new approach to MCE microvascular imaging. The major questions that now must be addressed are: (1) is the formation of microbubbles reproducible and safe, and (2) does this complicated approach have any major clinical advantage to state-of-the-art techniques already in practice today?
REFERENCES Author Manuscript Author Manuscript
1. Kaufmann BA, Wei K, Lindner JR. Contrast echocardiography. Curr Probl Cardiol. 2007; 32:51–96. [PubMed: 17208647] 2. Porter TR, Arena C, Sayyed S, Lof J, High RR, Xie F, Dayton PA. Targeted transthoracic acoustic activation of systemically administered nanodroplets to detect myocardial perfusion abnormalities. 2016; 9:e003770. 3. Sheeran PS, Luois S, Dayton PA, Matsunaga TO. Formulation and acoustic studies of a new phaseshift agent for diagnostic and therapeutic ultrasound. Langmuir : the ACS journal of surfaces and colloids. 2011; 27:10412–10420. [PubMed: 21744860] 4. Longmire M, Choyke PL, Kobayashi H. Clearance properties of nano-sized particles and molecules as imaging agents: Considerations and caveats. Nanomedicine. 2008; 3:703–717. [PubMed: 18817471] 5. Sirsi S, Feshitan J, Kwan J, Homma S, Borden M. Effect of microbubble size on fundamental mode high frequency ultrasound imaging in mice. Ultrasound Med Biol. 2010; 36:935–948. [PubMed: 20447755] 6. Ainslie MA, Leighton TG. Review of scattering and extinction cross-sections, damping factors, and resonance frequencies of a spherical gas bubble. The Journal of the Acoustical Society of America. 2011; 130:3184–3208. [PubMed: 22087992] 7. Sheeran PS, Matsunaga TO, Dayton PA. Phase-transition thresholds and vaporization phenomena for ultrasound phase-change nanoemulsions assessed via high-speed optical microscopy. Physics in medicine and biology. 2013; 58:4513–4534. [PubMed: 23760161] 8. Kaufmann BA, Lankford M, Behm CZ, French BA, Klibanov AL, Xu Y, Lindner JR. Highresolution myocardial perfusion imaging in mice with high-frequency echocardiographic detection of a depot contrast agent. J Am Soc Echocardiogr. 2007; 20:136–143. [PubMed: 17275698] 9. Lindner JR, Skyba DM, Goodman NC, Jayaweera AR, Kaul S. Changes in myocardial blood volume with graded coronary stenosis. Am J Physiol. 1997; 272:H567–575. [PubMed: 9038980] 10. 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; 97:473–483. [PubMed: 9490243]
Author Manuscript Circ Cardiovasc Imaging. Author manuscript; available in PMC 2017 January 01.
