3aBA1 – Ultrasound-Mediated Drug Targeting to Tumors: Revision of Paradigms Through Intravital Imaging

Natalya Rapoport – natasha.rapoport@utah.edu
Department of Bioengineering
University of Utah
36 S. Wasatch Dr., Room 3100
Salt Lake City, Utah 84112
USA

Popular version of paper 3aBA1, “Ultrasound-mediated drug targeting to tumors: Revision of paradigms through intravital imaging”
Presented Wednesday morning, May 25, 2016, 8:15 AM in Salon H
171st ASA Meeting, Salt Lake City

More than a century ago, Nobel Prize laureate Paul Ehrlich formulated the idea of a “magic bullet”. This is a virtual drug that hits its target while bypassing healthy tissues. No field of medicine could benefit more from the development of a “magic bullet” than cancer chemotherapy, which is complicated by severe side effects. For decades, the prospects of developing “magic bullets” remained elusive. During the last decade, progress in nanomedicine has enabled tumor-targeted delivery of anticancer drugs via their encapsulation in tiny carriers called nanoparticles. Nanoparticle tumor targeting is based on the “Achilles’ heels” of cancerous tumors – their poorly organized and leaky microvasculature. Due to their size, nanoparticles are not capable to penetrate through a tight healthy tissue vasculature. In contrast, nanoparticles penetrate through a leaky tumor microvasculature thus providing for localized accumulation in tumor tissue.  After tumor accumulation of drug-loaded nanoparticles, a drug should be released from the carrier to allow penetration into a site of action (usually located in a cell cytoplasm or nucleus). A local release of an encapsulated drug may be triggered by tumor-directed ultrasound; application of ultrasound has additional benefits: ultrasound enhances nanoparticle penetration through blood vessel walls (extravasation) as well as drug uptake (internalization) by tumor cells.

For decades, ultrasound has been used only as an imaging modality; the development of microbubbles as ultrasound contrast agents in early 2000s has revolutionized imaging. Recently, microbubbles have attracted attention as drug carriers and enhancers of drug and gene delivery. Microbubbles could have been ideal carriers for the ultrasound-mediated delivery of anticancer drugs.  Unfortunately, their micron-scale size does not allow effective extravasation from the tumor microvasculature into tumor tissue. In Dr. Rapoport’s lab, this problem has been solved by the development of nanoscale microbubble precursors, namely drug-loaded nanodroplets that converted into microbubbles under the action of ultrasound[1-6]. Nanodroplets comprised a liquid core formed by a perfluorocarbon compound and a two-layered drug-containing polymeric shell (Figure 1). An aqueous dispersion of nanodroplets is called nanoemulsion.

Rapoport 1 - Ultrasound-Mediated Drug Targeting

Figure 1. Schematic representation of a drug-loaded nanodroplet

A suggested mechanism of therapeutic action of drug-loaded perfluorocarbon nanoemulsions is discussed below [3, 5, 6]. A nanoscale size of droplets (ca. 250 nm) provides for their extravasation into a tumor tissue while bypassing normal tissues, which is a basis of tumor targeting. Upon nanodroplet tumor accumulation, tumor-directed ultrasound triggers nanodroplet conversion into microbubbles, which in turn triggers release of a nanodroplet-encapsulated drug. This is because in the process of the droplet-to-bubble conversion, particle volume increases about a hundred-fold, with a related decrease of a shell thickness. Microbubbles oscillate in the ultrasound field, resulting in a drug “ripping” off a thin microbubble shell (Figure 2). In addition, oscillating microbubbles enhance internalization of released drug by tumor cells.

Rapoport 2 - Ultrasound-Mediated Drug Targeting

Figure 2. Schematic representation of the mechanism of drug release from perfluorocarbon nanodroplets triggered by ultrasound-induced droplet-to-bubble conversion; PFC – perfluorocarbon

This tumor treatment modality has been tested in mice bearing breast, ovarian, or pancreatic cancerous tumors and has been proved very effective. Dramatic tumor regression and sometimes complete resolution was observed when optimal nanodroplet composition and ultrasound parameters were applied.

3A.Rapoport 3A 3B.Rapoport 3B 3C.Rapoport 3C

(Figure 3. A – Photographs of a mouse bearing a subcutaneously grown breast cancer tumor xenograft treated by four systemic injections of the nanodroplet-encapsulated anticancer drug paclitaxel (PTX) at a dose of 40 mg/kg as PTX. B – Photographs of a mouse bearing two ovarian carcinoma tumors (a) – immediately before and (b) – three weeks after the end of treatment; mouse was treated by four systemic injections of the nanodroplet-encapsulated PTX at a dose of 20 mg/kg as PTX; only the right tumor was sonicated. C – Photographs (a, c) and fluorescence images (b, d) of a mouse bearing fluorescent pancreatic tumor taken before (a, b) and three weeks after the one-time treatment with PTX-loaded nanodroplets at a dose of 40 mg/kg as PTX (c,d). The tumor was completely resolved and never recurred)[3, 4, 6].

In the current presentation, the proposed mechanism of a therapeutic action of drug-loaded, ultrasound-activated perfluorocarbon nanoemulsions has been tested using intravital laser fluorescence microscopy performed in collaboration with Dr. Brian O’Neill (then with Houston Methodist Research Institute, Houston, Texas) [2]. Fluorescently labeled nanocarrier particles (or a fluorescently labeled drug) were systemically injected though the tail vein to anesthetized live mice bearing subcutaneously grown pancreatic tumors. Nanocarrier and drug arrival and extravasation in the region of interest (i.e. normal or tumor tissue) were quantitatively monitored. Various drug nanocarriers in the following size hierarchy were tested: individual polymeric molecules; tiny micelles formed by a self-assembly of these molecules; nanodroplets formed from micelles. The results obtained confirmed the mechanism discussed above.

  • As expected, dramatic differences in the extravasation rates of nanoparticles were observed.
  • The extravsation of individual polymer molecules was extremely fast even in the normal (thigh muscle) tissue; In contrast, the extravasation of nanodroplets into the normal tissue was very slow. (Figure 4. A – Bright field image of the adipose and thigh muscle tissue. B,C – extravasation of individual molecules (B – 0 min; C – 10 min after injection); vasculature lost fluorescence while tissue fluorescence increased. D,E – extravasation of nanodroplets; blood vessel fluorescence was retained for an hour of observation (D – 30 min; E – 60 min after injection).
  • Nanodroplet extravasation into the tumor tissue was substantially faster than that into the normal tissue thus providing for effective nanodroplet tumor targeting.
  • Tumor-directed ultrasound significantly enhanced extravasation and tumor accumulation of both, micelles and nanodroplets (Figure 5). Also, pay attention to a very irregular tumor microvasculature, to be compared with that of a normal tissue shown in Figure 4.
  • The ultrasound effect on nanodroplets was 3-fold stronger than that on micelles thus making nanodroplets a better drug carriers for ultrasound-mediated drug delivery.
  • On a negative side, some premature drug release into the circulation that preceded tumor accumulation was observed. This proposes directions for a further improvement of nanoemulsion formulations.

Rapoport 5 - Ultrasound-Mediated Drug Targeting

Figure 5. Effect of ultrasound on the extravasation of Fluorescence of blood vessels dropped while that of the tumor tissue increased after ultrasound

3aBA – Using Ultrasound to Deliver Nanomedicine for the Treatment of Parkinson’s Disease

Richard J. Price – rprice@virginia.edu
University of Virginia
Box 800759, Health System
Charlottesville, VA 22908

Popular version of paper 3aBA
Presented Wednesday morning, May 25, 2016
171st ASA Meeting, Salt Lake City

Parkinson’s disease is characterized by the degeneration of nerve cells in the brain, often leading to poor balance, difficulties with walking, muscle pain and rigidity, tremors and involuntary movements, dementia, and memory loss. Fortunately, new gene therapy approaches for treating the root cause of the problem (i.e. neural cell degeneration) are beginning to show some pre-clinical success. These approaches involve introducing genes for neurotrophic factors [i.e. glial derived neurotrophic factor (GDNF)] into well-defined regions of the brain that are affected by neurodegeneration. Once the gene is introduced into the neural cells, the hope is that they will begin to manufacture the neurotrophic protein which, in turn, will halt neural degeneration. However, as currently implemented, there are significant weaknesses to such approaches. Foremost, the genes must be delivered by direct injection through a needle and/or infection-prone indwelling catheters. In addition to being highly invasive procedures, these direct injection approaches are unlikely to yield a homogeneous distribution of the gene in the target region of the brain. Indeed, due to both the fact that human gene therapy is in its genesis and that these current gene delivery procedures are highly invasive, only patients with very advanced disease will be considered candidates for treatment at first. This is unfortunate because, ideally, patients should be treated before significant degeneration occurs. Thus, the ultimate goal is to develop a new and minimally-invasive gene delivery approach for Parkinson’s that would incur minimal risk to the patient and therefore be safe enough to apply to healthy “early-stage” patients who are just beginning to exhibit symptoms.

Our proposed approach entails delivering non-viral neurotrophic gene-bearing nanocarriers to specific regions of the brain following their intravenous injection into the bloodstream. To achieve this, two physical barriers to gene delivery must be overcome. The first is the barrier offered by brain tissue itself, the so-called brain-tissue barrier (BTB). Our collaborators at Johns Hopkins University have developed a new technology that allows nanoparticles to diffuse easily through the BTB. These so-called “brain-penetrating nanoparticles” exhibit uniform, long-lasting, and effective delivery. The second barrier to delivery is the blood-brain barrier (BBB), the essentially impenetrable membrane created by brain capillaries that separates the bloodstream from brain tissue. The Price lab at the University of Virginia has been studying how the BBB may be opened in a site selective manner for targeted drug and gene delivery. In essence, they have shown that applying focused ultrasound energy to the brain after the injection of micron-sized gas bubbles (FDA approved for other applications) can open the BBB (Figure 1). Of particular importance to this project, the Price group has demonstrated that opening the BBB with this targeted technology permits the delivery of brain-penetrating nanoparticles (fabricated in the Hanes lab) from the bloodstream to the tissue. The nanoparticles are transported by diffusion and convection to the brain and distribute evenly throughout, yielding homogeneous delivery without an invasive transcranial injection.

Price Figure 1 - Parkinson’s Disease

Figure 1. Transcranial focused ultrasound achieves non-invasive, safe, repeated and targeted blood-brain barrier disruption, leading to improved drug or gene delivery.

Advancing this concept to the clinic as a treatment for Parkinson’s disease will require testing the efficacy of the approach in a small animal model of neurodegeneration. Here, we first delivered non-viral reporter gene nanoparticles to rat brain using focused ultrasound, resulting in robust dose-dependent gene expression, only in the region exposed to ultrasound, through day 28. We also measured a transfection efficiency (i.e. the percentage of cells expressing the delivered gene) at > 40%. Toxicity was not evident. We then tested whether the approach had therapeutic potential for treating Parkinson’s disease by delivering neurotrophic (GDNF) gene nanoparticles to the striatum of Parkinson’s rats. MR images of BBB opening with focused ultrasound in the striatum are shown in Figure 2. For treated rats, motor impairment tests (apomorphine-induced rotation and cylinder) revealed significant improvement and dopaminergic neuron density was fully restored in key brain structures (i.e. striatum and substantia nigra pars compacta). We conclude that image-guided nanoparticle delivery with focused ultrasound is a safe and non-invasive strategy for brain transfection that has potential to be translated into a non-invasive clinical treatment for Parkinson’s disease.

Price Figure 2

Figure 2. Left: MR image showing structure of the striatum (outlined in yellow), which is the brain region targeted for treatment. Right: MR image of the striatum after treatment with focused ultrasound. The 4 bright spots show where the BBB has been opened in the striatum, allowing for the delivery of gene nanoparticles.

2aMU4 – Yelling vs. Screaming in Operatic and Rock Singing

Lisa Popeil – lisa@popeil.com
Voiceworks®
14431 Ventura Blvd #200
Sherman Oaks, CA 91423

Popular version of paper 2aMU4
Presented Tuesday morning, May 24, 2016

There exist a number of ways the human vocal folds can vibrate which create unique sounds used in singing.  The two most common vibrational patterns of the vocal folds are commonly called “chest voice” and “head voice”, with chest voice sounding like speaking or yelling and head voice sounding more flute-like or like screaming on high pitches.  In the operatic singing tradition, men sing primarily in chest voice while women sing primarily in their head voice.  However, in rock singing, men often emit high screams using their head voice while female rock singers use almost exclusively their chest voice for high notes.

Vocal fold vibrational pattern differences are only a part of the story though, since the shaping of the throat, mouth and nose (the vocal tract) play a large part in the perception of the final sound.  That means that head voice can be made to “sound” like chest voice on high screams using vocal tract shaping and only the most experienced listener can determine if the vocal register used was chest or head voice.

Using spectrographic analysis, differences and similarities between operatic and rock singers can be seen.  One similarity between the two is the heightened output of a resonance commonly called “ring”.  This resonance, when amplified by vocal tract shaping, creates a piercing sound that’s perceived by the listener as extremely loud. The amplified ring harmonics can be seen in the 3,000 Hz band in both the male opera sample and in rock singing samples:

MALE OPERA – HIGH B (B4…494 Hz) CHEST VOICEPopeil1 Check Voice SingingFigure 1 MALE ROCK – HIGH E (E5…659 Hz) CHEST VOICEPopeil 2 Chest voice singingFigure 2 MALE ROCK – HIGH G (G5…784 Hz)    HEAD VOICEPopeil 3 Head voice singingFigure 3

Though each of these three male singers exhibit a unique frequency signature and whether singing in chest or head voice, each singer is using the amplified ring strategy in the 3,000Hz range amplify their sound and create excitement.

2aMU5 – Do people find vocal fry in popular music expressive?

Mackenzie Parrott – mackenzie.lanae@gmail.com
John Nix – john.nix@utsa.edu

Popular version of paper 2aMU5, “Listener Ratings of Singer Expressivity in Musical Performance.”
Presented Tuesday, May 24, 2016, 10:20-10:35 am, Salon B/C, ASA meeting, Salt Lake City

Vocal fry is the lowest register of the human voice.  Its distinct sound is characterized by a low rumble interspersed with uneven popping and crackling.  The use of fry as a vocal mannerism is becoming increasingly common in American speech, fueling discussion about the implications of its use and how listeners perceive the speaker [1].  Previous studies have suggested that listeners find vocal fry to be generally unpleasant in women’s speech, but associate it with positive characteristics in men’s speech [2].

As it has become more prevalent, fry has perhaps not surprisingly found its place in many commercial song styles as well.  Many singers are implementing fry as a stylistic device at the onset or offset of a sung tone.  This can be found very readily in popular musical styles, presumably to impact and amplify the emotion that the performer is attempting to convey.

Researchers at the University of Texas at San Antonio conducted a survey to analyze whether listeners’ ratings of a singer’s expressivity in musical samples in two contemporary commercial styles (pop and country) were affected by the presence of vocal fry, and to see if there was a difference in listener ratings according to the singer’s gender.  A male and a female singer recorded musical samples for the study in a noise reduction booth.  As can be seen in the table below, the singers were asked to sing most of the musical selections twice, once using vocal fry at phrase onsets, and once without fry, while maintaining the same vocal quality, tempo, dynamics, and stylization.  Some samples were presented more than one time in the survey portion of the study to test listener reliability.

Song Singer Gender Vocal Mode
(Hit Me) Baby One More Time Female Fry Only
If I Die Young Female With and Without Fry
National Anthem Female With and Without Fry
Thinking Out Loud Male Without Fry Only
Amarillo By Morning Male With and Without Fry
National Anthem Male With and Without Fry

Across all listener ratings of all the songs, the recordings which included vocal fry were rated as being only slightly more expressive than the recordings which contained no vocal fry.  When comparing the use of fry between the male and female singer, there were some differences between the genders.  The listeners rated the samples where the female singer used vocal fry higher (e.g., more expressive) than those without fry, which was surprising considering the negative association with women using vocal fry in speech.  Conversely, the listeners rated the male samples without fry as being more expressive than those with fry. Part of this preference pattern may have also been an indication of the singer; the male singer was much more experienced with pop styles than the female singer, who is primarily classically trained.  The overall expressivity ratings for the male singer were higher than those of the female singer by a statistically significant margin.

There were also listener rating trends between the differing age groups of participants.  Younger listeners drove the gap of preference between the female singer’s performances with fry versus non-fry and the male singer’s performances without fry versus with fry further apart.  Presumably they are more tuned into stylistic norms of current pop singers.  However, this could also imply a gender bias in younger listeners.  The older listener groups rated the mean expressivity of the performers as being lower than the younger listener groups.  Since most of the songs that we sampled are fairly recent in production, this may indicate a generational trend in preference.  Perhaps listeners rate the style of vocal production that is most similar to what they listened to during their young adult years as the most expressive style of singing. These findings have raised many questions for further studies about vocal fry in pop and country music.

 

  1. Anderson, R.C., Klofstad, C.A., Mayew, W.J., Venkatachalam, M. “Vocal Fry May Undermine the Success of Young Women in the Labor Market. “ PLoS ONE, 2014. 9(5): e97506. doi:10.1371/journal.pone.0097506.
  2. Yuasa, I. P. “Creaky Voice: A New Feminine Voice Quality for Young Urban-Oriented Upwardly Mobile American Women.” American Speech, 2010. 85(3): 315-337.

2aPA8 – Taming Tornadoes: Controlled Trapping and Rotation with Acoustic Vortices

Asier Marzo – amarzo@hotmail.com
Mihai Caleap
Bruce Drinkwater

Bristol University
Senate House, Tyndall Ave,
Bristol, United Kingdom

Popular version of paper 2aPA8, “Taming tornadoes: Controlling orbits inside acoustic vortex traps”
Presented Tuesday afternoon, May 24, 2016, 11:05 AM, Salon H
171st ASA Meeting Salt Lake City

Tractor beams are mysterious beams that have the ability to attract objects towards the source of the emission (Figure 1). These beams have attracted the attention of both scientists and sci-fi fans. For instance, it is quite an iconic device in Star Wars or Star Trek where it is used by big spaceships to trap and capture smaller objects.

Figure-01

Figure 1. A sonic tractor beam working on air.

In the scientific community, they have been studied theoretically for decades and in 2014, a tractor beam made with light was realized [1]. It used the energy of the photons bouncing on a microsphere to keep it trapped laterally and at the same time heated the back of the sphere with different light patterns to pull it towards the laser source. The sphere had a diameter of 50 micrometres, was made of glass and coated with gold.

A tractor beam made with light can only manipulate very small particles and made of specific materials. Making a tractor beam which uses mechanical waves (i.e. sound or ultrasound) would enable the trapping of a much wider range of particle sizes and allow almost any combination of particle and host fluid materials, for example drug delivery agents within the human body.

Recently, it has been proven experimentally that a Vortex beam can act as a tractor beam both in air [2] and in water [3]. A Vortex beam (such as a first order Bessel beam) is analogous to a tornado of sound which is hollow in the middle and spirals about a central axis, the particles get trapped in the calm eye of the tornado (Figure 2).

Figure-02 - Acoustic Vortices

Figure 2. Intensity iso-surface of an Acoustic Vortex. 54 ultrasonic speakers emitting at 40kHz arranged in a hemisphere (see [2] for fuller details) create an acoustic vortex that traps the particle in the middle.

The problem is, that only very small particles are stably trapped inside the vortex. As the particles get bigger, they start to spin and orbit until being ejected (Figure 3). As in a tornado, only the small particles remain within the vortex whereas the larger ones get ejected.

Figure-03

Figure 3. Particle behaviour depending on its size: a small particle is trapped (a), a middle particle orbits (b) and big particles gets ejected (c).

Here we show that, contrary to a tornado, we can change the direction of an acoustic vortex thousands of times per second. In our paper, we prove that by rapidly switching the direction of the acoustic vortex it is possible to produce stable trapping of particles of various sizes. Furthermore, by adjusting the proportion of time that each vortex direction is emitted, the spinning speed of the particle can be controlled (Figure 4).

Figure-04 - Acoustic Vortices

Figure 4. Taming the vortex: a) the vortex rotates all the time in the same direction and this rotation is transferred to the particle. b) the vortex switches direction and thus the angular momentum is completely or partially cancelled, providing rotational control.

The ability to levitate and controllably rotate inside acoustic vortices particles such as liquids, crystals or even living cells enables new possibilities and processes for a variety of disciplines.

References

  1. Shvedov, V., Davoyan, A. R., Hnatovsky, C., Engheta, N., & Krolikowski, W. (2014). A long-range polarization-controlled optical tractor beam. Nature Photonics, 8(11), 846-850.
  2. Marzo, A., Seah, S. A., Drinkwater, B. W., Sahoo, D. R., Long, B., & Subramanian, S. (2015). Holographic acoustic elements for manipulation of levitated objects. Nature communications, 6.
  3. Baresch, D., Thomas, J. L., & Marchiano, R. (2016). Observation of a single-beam gradient force acoustical trap for elastic particles: acoustical tweezers. Physical Review Letters, 116(2), 024301.