Introduction

Acoustic Droplet Vaporization (ADV) is a phase change phenomenon in which the liquid state, in the form of droplets, is converted to gas as a result of bursts in the excited ultrasound field. Having a wide range of medical applications, ADV has drawn considerable attention in imaging [1], diagnosis and critical medical treatment [2]. Therefore, benefitting from its broad potentials, with the consideration of its capability in localized noninvasive energy exposure, it is possible to utilize its effect in different medical applications from targeted drug delivery [3] to embolotherapy [4].

Apart from the droplet characterization and ADV effectiveness on the applied region, the physics of ADV and particularly the ultrasound analysis is an essential parameter in the initiation of the vaporization. This part, which is related to acoustic wave physics, implies that ADV is mostly dependent on ultrasound pressure, frequency and temperature. In this sense, Miles et al. [5] tried to find incident negative pressure - called as ADV threshold- which is necessary for the induction of nucleation. It was successfully shown that the negative pressure required for the nucleation prior to collapse can be determined via perturbation analysis of a compressible inviscid flow around a droplet for various frequencies and diameters. In addition, the fluid medium which constitutes the droplet emulsion and the surrounding fluid constructs a significant field within ADV. In this regard, there are many studies which illustrated that the diameter of the droplets subjected to the acoustic waves undergoes a significant expansion of 5 to 6 times of their regular sizes [6-8].

In this study, a new type of pickering stabilized perfluorodroplets (PFC) was examined under the effect of the different acoustic parameters to evaluate its potential in the acoustic droplet vaporization process. To assess the pressure effects on the stabilized droplets, the acoustic power within the ultrasound tests was varied and the phase trasnition was characterized according to the experimental conditions. Opticell® was utilized as the transparent device to visualize the droplets, which were exposed to the acoustic waves with the aid of the microscope and multi-well microplate.

Methods

Materials and emulsion preparation

Perfluoropentane (PFC5) was purchased from Apollo Scientific (City, U.K.). Bleached sulfite pulp (from Nordic Paper Seffle AB, Sweden) was used in the production of the cationic cellulose nanofibers (CNFs). The CNF suspension (1.32 wt%) were prepared as described previously [9]. The CNFs had a dimension of 3.9 ± 0.8 nm in width and a length in the micrometer range. The amount of cationic groups was 0.13 mmol per g fiber, obtained from conductometric titration [9]. A suspension of CNF (0.28 wt%) was prepared by diluting the stock CNF with MilliQ-water (pH of diluted CNF suspension was 9.5). The suspension was treated with ultra-sonication at amplitude of 90% for 180 s (Sonics, Vibracell W750). The suspension was brought to room temperature. An amount of 36 g of the 0.28 wt% CNF suspension was mixed with 1 g of PFC5. The mixture was sonicated for 60s at an amplitude of 80% (under ice-cooling) to obtain the CNF-stabilized PFC5 droplets.

The protocol for the acoustic tests

100 μL of CNF-stabilized PFC5 droplets were added to 1900 μL of deionized water in order to prepare the solution which were exposed to the ultrasound waves. The droplet sample, diluted 1:19 in distilled water was introduced to the Opticell® and the acoustic waves at a fixed frequency and different powers were applied to the trageted area inside the Opticell® which is located inside a water bath. The ultrasound triggered sample then was placed under a 20X magnification objective of upright transmitted light microscope (ECLIPSE Ci-S, Nikon, Tokyo, Japan). 

The acoustic tests were performed using high-power tone burst pulser-receiver (SNAP Mark IV,  Ritec, Inc., Warwick, RI, USA) equipped with a transducer (V382-SU Olympus NDT, Waltham, MA ) operating at the frequency of 3.5 MHz. The emulsion of CNF-stabilized PFC5 droplets were exposed to the power range which has the acsending trend from -30 to 0 dB at the given frequency. To investigate the droplet size variations at each power between, the droplets were collected inside the Opticell® and the droplet diameter was measured with the aid of the ImageJ software (version 1.50b, National institutes of health, USA) to determine the concentration and size distribution. The Gaussian distribution is ploted with mean value and standad deviation recover from the experimental data. An in-house image edge detection MATLAB™ script (MathWorks Inc., Natick, MA) were applied to analyze the images obtained from the microscope and provides the size and volume distributions.

Results

The size of PFP droplets is an important parameter to controll in the therapeutic applications. Here, a new type of Pickering stabilized perfluorodroplets were prepared where the PFP/water interface was stabilized with cellulose nanofibers (CNF) and the size of the droplets could easily be controlled by varying the amount of CNF added.  The resulting droplets were investigated using a single crystal transducer. Apart from the medical applications, controlling the droplet size is important from droplet dynamics point of view, becausethe interfacial energy is crucial in the assumption of the critical nucleus radius. Therefore, it is possible to estimate the negative peak pressure required for the phase transition once the droplet is controlled and interfacial energy deposited inside and on the surface of the droplet are balanced.

According to the results in Figure 1, there is an appreciable rise of the size of the droplets after ultrasound waves exposure, particularly at -8 dB power. The experiments were performed for 30 seconds at different powers ranging from -30 to 0 dB, while the frequency was kept constant at 3.5 MHz, burst width in cycles was selected as 12 and repetition rate was set to 100. Images included in Figure 1 demonstrate major transitions in the intervals at -16, -8 and 0 dB. As shown in this figure, the droplet size increased with the power rise and more bubbles with bigger sizes appears at higher powers. This outcome implies the significant role of the applied frequency and power on the phase shift and subsequent mechanisms as a result of the acoustic wave exposure on the new nontoxic and incompatible droplet type.

Figure 2 shows the average number of droplets and volume distribution at the corresponding powers to the Figure 1. It is shown that while the average diameter of the droplets is around 3.5 µm, the generated bubbles, as a result of the ADV, reaches up to 15 µm at the highest possible power. For each set of experiment (corresponding to a given power) 32 images were taken, thus, to reduce the errors and obtain the standard deviation (approximately 0.8 for all the cases), the presented diagrams for the droplet distributions exhibits the mean value for all of the acquired images. Therefore, it is shown that the droplet emulsion exhibited in NO US in Figure 2, which shows the regular view and distribution range of the CNF-stabilized PFC5 droplets at the room temperature, experiences ADV process with the diameter rise of about 5 times at the highest power when the frequency is fixed at 3.5 MHz.

Conclusions

The results show that there is appreciable rise on the size of the droplets after ultrasound waves exposure at a fixed frequency. Acoustic droplet vaporization (ADV) was illustrated at different powers for CNF-stabilized PFC5 droplets as a new class of pickering stabilized perfluorodroplets with the increase in the size of the droplets and following phase trasition to bubbles. Diameter increase of 5 times were obtained after the ultrasound exposure indicating the efficiency of the suggested droplets for the ADV process and therapeutic applications.   

References

[1] Arena CB, Novell A, Sheeran PS, Puett C, Moyer LC, Dayton PA, Dual-Frequency Acoustic Droplet Vaporization Detection for Medical Imaging 2015, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control, 62: 9.

[2] Kripfgans OD, Fowlkes JB, Miller DL, Eldevik OP, Carson PL, Acoustic droplet vaporization for therapeutic and diagnostic applications 2000, Ultrasound Med. Biol, 26:1177–1189.

[3] Kang ST, Yeh CK, Intracellular Acoustic Droplet Vaporization in a Single Peritoneal Macrophage for Drug Delivery Applications 2011, Langmuir, 27:13183–13188.

[4] Zhu M, Jiang L, Fabiilli ML, Zhang A, Fowlkes JB, Xu LX, Treatment of murine tumors using acoustic droplet vaporization-enhanced high intensity focused 2013, Ultrasound Phys. Med. Biol, 58:6179–6191.

[5] Miles CJ, Doering CR, Kripfgans OD, Nucleation pressure threshold in acoustic droplet vaporization 2016, Journal of Applied Physics, 120:034903.

[6] Sheeran PS, Wong VP, Luois S, McFarland RJ, Ross WD, Feingold S, Matsunaga TO, Dayton PA, Decafluorobutane as a phase-change contrast agent for low-energy extravascular ultrasonic imaging 2011, Ultrasound Med. Biol, 37:1518–1530.

[7] Kripfgans OD, Fowlkes JB, Miller DL, Eldevik OP, Carson PL, Acoustic droplet vaporization for therapeutic and diagnostic applications 2000, Ultrasound Med. Biol, 26:1177–1189.

[8] Kang S, Huang Y, Yeh C, Characterization of acoustic droplet vaporization for control of bubble generation under flow conditions 2014, Ultrasound Med. Biol, 40:551–561.

[9] Svagan AJ, Benjamins JW, Al-Ansari Z, Shalom DB, Müllertz A, Wågberg L, Löbmann K, Solid cellulose nanofiber based foams–towards facile design of sustained drug delivery systems 2016, J. Control Release, 244:74–82 (Part A).

Acoustic droplet vaporization (ADV) is the physical process in which liquid undergoes phase transition to gas after exposure to a pressure amplitude above a certain threshold. In recent years, new techniques in ultrasound diagnostics and therapeutics have been developed which utilize microformulations with various physical and chemical properties. The purpose of this review is to give the reader a general idea on how ADV can be implemented for the existing biomedical applications of droplet vaporization. In this regard, the recent developments in ultrasound therapy which shed light on the ADV are considered. Modern designs of capsules and nanodroplets (NDs) are shown, and the material choices and their implications for function are discussed. The influence of the physical properties of the induced acoustic field, the surrounding medium, and thermophysical effects on the vaporization are presented. Lastly, current challenges and potential future applications towards the implementation of the therapeutic droplets are discussed.

Acoustophoresis, the ability to acoustically manipulate particles and cells inside a microfluidic channel, is a critical enabling technology for cell-sorting applications. However, one of the major impediments for routine use of acoustophoresis at clinical laboratory has been the reliance on the inherent physical properties of cells for separation. Here, we present a microfluidic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method that rely on the specific binding of target cells to microbubbles conjugated with specific antibodies on their surface for continuous cell separation using ultrasonic standing wave. In acoustophoresis, cells being positive acoustic contrast particles migrate to pressure nodes. On the contrary we show that air-filled polymer-shelled microbubbles being strong negative acoustic contrast particles migrate to pressure antinodes at acoustic pressure amplitudes as low as 60 kPa. As a proof of principle, using the BAACS strategy, we demonstrate the separation of cancer cell line in a suspension with better than 75% efficiency. Moreover, 100% of the microbubble-cell conjugates migrated to the anti-node. Hence a better upstream affinity-capture has the potential to provide higher sorting efficiency. The BAACS technique may potentially provide a simplistic approach for similar sized selective isolation of cells, and is suited for applications in point of care.

Polymer shelled gas microbubbles (MBs) are used to sort cells in a microfluidic chip under acoustic standing waves (SW). When particles are subjected to SW based on their acoustic contrast factor (ACF) they migrate to nodes (positive contrast factor particles; PACP) or antinodes (negative acoustic contrast particles; NACP)[1]. We have bounded functionalized MBs with cells such that, they can be selectively migrated to antinodes under SW and sorted from unbounded cell both in no flow and flow conditions. Here we demonstrate acoustic mediated microbubble tagged cell sorting with 75% efficiency.

Numerous studies have been carried out in the past few decades to investigate the radial oscillations of encapsulated microbubbles (MBs). Nonspherical oscillations also have gained attention, being unavoidable in actual applications of these bubbles. The present paper is intended to describe the nature of resonance trends of such spherical and nonspherical modes of a thick encapsulated MB filled with air and suspended in water. The shell material is assumed to be linear viscoelastic and quasi-incompressible. The considered isotropic and spherically isotropic material parametric range is limited to thick polymer shelled MBs. For the case of an isotropic material, shell viscosity has a major influence on the fundamental modes with meridional wave number n = 0, 4, especially for thicker bubbles, unlike for the case of the spherically isotropic material case considered, where the viscosity has very little influence. For most of the parametric range, n = 2, 3 modes are underdamped and their frequency is found to be lower than the n = 0, 4 modes, for both material cases. An interesting case is found for a spherically isotropic quasiincompressible material case, where the first few nonspherical mode resonances are very close to radial mode resonance frequency.

Objective

Quantification of the myocardial perfusion by contrast echocardiography (CEC) remains a challenge. Existing imaging phantoms used to evaluate the performance of ultrasound scanners do not comply with perfusion basics in the myocardium, where perfusion and motion are inherently coupled.

Methods

To contribute towards an improvement, we developed a CEC perfusion imaging platform based on isolated rat heart coupled to the ultrasound scanner. Perfusion was assessed using three different types of contrast agent: dextran-based Promiten^®, phospholipid-shelled SonoVue^®, and polymer-shelled MB-pH5-RT. The myocardial video-intensity was monitored over time from contrast administration to peak and two characteristic constants were calculated using exponential fit (A representing capillary volume and b representing inflow velocity).

Results

Acquired experimental evidence demonstrates that the application of all three types of contrast agent allow ultrasonic estimation of myocardial perfusion in the isolated rat heart. Video-intensity maps show that an increase in contrast concentration increases the late plateau values, A, mimicking increased capillary volume. Estimated values of the flow, proportional to Axb, increase when the pressure of the perfusate column increases from 80 to 110 cm of water. This finding is in agreement with the true values of the coronary flow increase measured by the flowmeter attached to the aortic cannula.

Conclusions

The described CEC perfusion imaging platform holds promise for standardized evaluation and optimization of ultrasound contrast perfusion imaging where real time inflow curves at low acoustic power semi-quantitatively reflect coronary flow.

The current study describes novel multifunctional polymer-shelled microbubbles (MBs) loaded with nitric oxide (NO) for integrated therapeutic and diagnostic applications, i.e. theranostics, of myocardial ischemia. We used gas filled MBs with an average diameter of 4 µm stabilized by a biocompatible shell of poly(vinyl)alcohol. In vitro acoustic tests showed a sufficient enhancement of the backscattered power (20 dB) acquired from the MBs suspension. The values of attenuation coefficient (0.8 dB/cm MHz) and phase velocities (1517 m/s) were comparable to those reported for the soft tissue. Moreover, polymer MBs demonstrate increased stability compared to clinically approved contrast agents with fracture threshold of about 900 kPa. In vitro chemiluminescence measurements demonstrated that dry powder of NO-loaded MBs releases its gas content in about 2 hours following an exponential decay profile with an exponential time constant equal 36 min. The application of high power ultrasound pulse (MI=1.2) on the MBs resuspended in saline decreases the exponential time constant from 55 to 4 min in air saturated solution and from 17 to 10 min in degased solution. Thus, ultrasound-triggered release of NO is achieved. Cytotoxicity tests indicate that phagocytosis of the MBs by macrophages starts within 6 to 8 hours. This is suitable time for initial diagnostics, treatment and monitoring of the therapeutic effect using single injection of the proposed multifunctional MBs.

Combinations of microbubbles (MBs) and superparamagnetic iron oxide nanoparticles (SPIONs) are used to fabricate dual contrast agents for ultrasound and MRI. This study examines the viscoelastic and oscillation characteristics of two MB types that are manufactured with SPIONs and either anchored chemically on the surface (MBs-chem) or physically embedded (MBs-phys) into a polymer shell. A linearized Church model was employed to simultaneously fit attenuation coefficients and phase velocity spectra that were acquired experimentally. The model predicted lower viscoelastic modulus values, undamped resonance frequencies and total damping ratios for MBs-chem. MBs-chem had a resonance frequency of approximately 13 MHz and a damping ratio of approximately 0.9; thus, MBs-chem can potentially be used as a conventional ultrasound contrast agent with the combined functionality of MRI detection. In contrast, MBs-phys had a resonance frequency and damping of 28 MHz and 1.2, respectively, and requires further modification of clinically available contrast pulse sequences to be visualized.

Polymer-shelled magnetic microbubbles have great potential as hybrid contrast agents for ultrasound and magnetic resonance imaging. In this work, we studied US/MRI contrast agents based on air-filled poly(vinyl alcohol)-shelled microbubbles combined with superparamagnetic iron oxide nanoparticles (SPIONs). The SPIONs are integrated either physically or chemically into the polymeric shell of the microbubbles (MBs). As a result, two different designs of a hybrid contrast agent are obtained. With the physical approach, SPIONs are embedded inside the polymeric shell and with the chemical approach SPIONs are covalently linked to the shell surface. The structural design of hybrid probes is important, because it strongly determines the contrast agent's response in the considered imaging methods. In particular, we were interested how structural differences affect the shell's mechanical properties, which play a key role for the MBs' US imaging performance. Therefore, we thoroughly characterized the MBs' geometric features and investigated low-frequency mechanics by using atomic force microscopy (AFM) and high-frequency mechanics by using acoustic tests. Thus, we were able to quantify the impact of the used SPIONs integration method on the shell's elastic modulus, shear modulus and shear viscosity. In summary, the suggested approach contributes to an improved understanding of structure-property relations in US-active hybrid contrast agents and thus provides the basis for their sustainable development and optimization.

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.