Acoustic droplet vaporization-mediated dissolved oxygen scavenging in blood-mimicking fluids, plasma, and blood

Accepted Manuscript Acoustic droplet vaporization-mediated dissolved oxygen scavenging in bloodmimicking fluids, plasma, and blood Karla P. Mercado-Shekhar, Haili Su, Deepak S. Kalaikadal, John N. Lorenz, Raj M. Manglik, Christy K. Holland, Andrew N. Redington, Kevin J. Haworth PII: DOI: Reference:

S1350-4177(18)31507-4 https://doi.org/10.1016/j.ultsonch.2019.03.029 ULTSON 4538

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

3 October 2018 20 December 2018 27 March 2019

Please cite this article as: K.P. Mercado-Shekhar, H. Su, D.S. Kalaikadal, J.N. Lorenz, R.M. Manglik, C.K. Holland, A.N. Redington, K.J. Haworth, Acoustic droplet vaporization-mediated dissolved oxygen scavenging in bloodmimicking fluids, plasma, and blood, Ultrasonics Sonochemistry (2019), doi: https://doi.org/10.1016/j.ultsonch. 2019.03.029

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Acoustic droplet vaporization-mediated dissolved oxygen scavenging in bloodmimicking fluids, plasma, and blood Karla P. Mercado-Shekhar,a Haili Su,a Deepak S. Kalaikadal,b John N. Lorenz,c Raj M. Manglik,b Christy K. Holland,a,d Andrew N. Redington,e and Kevin J. Haworth a,d,*

a

Department of Internal Medicine, University of Cincinnati, Cincinnati, Ohio,

U.S.A. b

Department of Mechanical and Materials Engineering, University of Cincinnati,

Cincinnati, , Ohio, U.S.A. c

Department of Pharmacology and Systems Physiology, University of Cincinnati,

Cincinnati, Ohio, U.S.A. d Department

of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio,

U.S.A. e

Division of Cardiology, The Heart Institute, Cincinnati Children’s Hospital

Medical Center, Cincinnati, Ohio, U.S.A. * Corresponding author

Address for correspondence: Kevin J. Haworth, PhD

1

University of Cincinnati, 231 Albert Sabin Way. Cardiovascular Research Center, Rm 3939 Cincinnati, Ohio 45267-0586 Phone: 1.513.558.3536 Email: [email protected]

2

Abstract Acoustic droplet vaporization (ADV) has been shown to reduce the partial pressure of oxygen (PO2) in a fluid. The goals of this study were three-fold: 1) to determine the ADV pressure amplitude threshold in fluids that had physiologically relevant values for surface tension, protein concentration, and viscosity; 2) to assess whether these parameters and fluid mixing affect ADV-mediated PO2 reduction; and 3) to assess the feasibility of ADV-mediated PO2 reduction in plasma and whole blood. In vitro ADV experiments were conducted using perfluoropentane droplets (number

density:

5106±0.2106

/mL)

dispersed

in

fluids

(saline,

polyvinylpyrrolidone solutions, porcine plasma, or porcine whole blood) that had a physiological range of surface tensions (62–68 mN/m), protein concentrations (0 and 68.7 mg/mL), and viscosities (0.7–4 cP). Droplets were exposed to pulsed ultrasound (5 MHz, 4.25 MPa peak negative pressure) while passing through a 37 °C flow system with inline PO2 sensors. In select experiments, the fluid also passed through mixing channels after ultrasound exposure. Our results revealed that the ADV pressure thresholds were the same for all fluids. Surface tension and protein concentration had no effect on PO2 reduction. Increasing viscosity attenuated PO2 reduction. However, the attenuated effect was absent after fluid mixing. Furthermore, ADV-mediated PO2 reduction in whole blood (30.8 ± 3.2 mmHg) was less than that in a polyvinylpyrrolidone solution (40.2 ± 2.1 mmHg) with equal

3

viscosity. These findings should be considered when planning clinical studies of ADV-mediated PO2 reduction and other biomedical applications of ADV.

4

Highlights:  Physiological surface tension had a negligible effect on ADV-mediated PO2 reduction.  Fluid protein concentration was uncorrelated with PO2 reduction.  Viscosity had a transient effect on PO2 reduction.  Fluid mixing accelerated PO2 reduction at physiologic viscosities.  Reduction in the PO2 in whole blood was demonstrated.

Keywords: ultrasound-mediated phase transition; physiological fluid properties; diffusion; fluid mixing; dissolved oxygen scavenging; whole blood

Abbreviations ADV: Acoustic droplet vaporization PBS: Phosphate-buffered saline PVP: Polyvinylpyrrolidone PFP: Perfluoropentane BSA: Bovine serum albumin PDI: Polydispersity index MI: Mechanical index EVA: Ethyl vinyl acetate 5

PE: Polyethylene PO2: Partial pressure of oxygen PN2: Partial pressure of nitrogen PCO2: Partial pressure of carbon dioxide SaO2: Oxygen saturation of hemoglobin

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1. Introduction Acoustic droplet vaporization (ADV) is the phase transition of submicronand micron-sized liquid perfluorocarbon droplets into gas microbubbles upon exposure to sufficient acoustic energy [1]. ADV has been investigated for both diagnostic and therapeutic ultrasound applications, including contrast-enhanced imaging [1–7], aberration correction [8], embolotherapy [9,10], ultrasound ablation [11–15], and targeted drug delivery [16–22]. The volume of perfluorocarbon gas microbubbles formed by ADV is nearly 125-fold higher than the volume of the originating microdroplets [1], and after ADV, the microbubbles continue to grow due to ingassing [1,3,23,24]. Recently, Radhakrishnan and colleagues demonstrated that the phase transition of perfluoropentane microdroplets by ADV reduced the partial pressure of oxygen (PO2) in phosphate-buffered saline (PBS) [25]. The volumetric expansion of the microbubbles caused a PO2 difference in the microbubbles and surrounding fluid. Thereafter, the microbubbles acted as potent gas sinks capable of carrying more oxygen than an equal volume of the surrounding aqueous fluid [26]. Perfluoropentane and oxygen diffused across the microbubble interface (in opposite directions). However, because the Ostwald coefficient of perfluoropentane in water (1.2x10-4) was assumed to be two orders of magnitude less than that in oxygen (2.8

7

x10-2) [27], substantially more oxygen was expected to enter the microbubbles than perfluoropentane to diffuse into the surrounding fluid. As a step towards clinical translation of ADV-mediated PO2 reduction, experiments with biological fluids, such as plasma and whole blood, are required because the physical properties (e.g., surface tension and viscosity) of these fluids are different from those of saline [28]. These fluid properties have been shown to affect the ADV process [3,23,29,30]. Sheeran et al. [3] found that an increase in the interfacial surface tension from 30 mN/m to 51 mN/m reduced the radial expansion factor of droplets. Qamar et al. [30] showed numerically that the expansion factor of perfluoropentane droplets decreased as the surface tension increased from 60 mN/m to 500 mN/m. These effects were more pronounced as the initial droplet sizes decreased. Qamar et al. [30] also simulated ADV in fluids of different viscosities ranging from 0.6 cP to 4 cP and found that higher viscosities did not affect the final size of the bubble. However, increasing viscosity delayed the time to reach the final size [30]. Furthermore, Kang et al. [23] demonstrated experimentally that perfluoropentane droplets diluted in porcine blood plasma (1.4 cP to 2 cP) exhibited a slower growth rate and had smaller final bubble sizes compared to those in saline (0.7 cP). Additionally, Fabiilli et al. [29] demonstrated that increasing fluid viscosity increased the pressure amplitude threshold of ADV, but surface tension did not have an effect. The exact mechanisms for these observations have not been elucidated.

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When ADV occurs within a small focal volume of a viscous fluid, a delay in dissolved oxygen scavenging outside that focal volume may occur [31]. Fluid mixing could facilitate dissolved oxygen scavenging by reducing the diffusion path length between oxygen (O2) molecules from outside the focal volume and ADV microbubbles. Additionally, microbubbles formed by ADV of bovine serum albumin-coated droplets have incomplete shells [24]. Thus, proteins in the surrounding fluid (e.g., plasma proteins in whole blood) could adsorb onto the free gas-fluid interfaces, affecting the surface tension [32], and consequently, gas diffusion across the microbubble shell [33–35]. Moreover, the protein molecules in solution could obstruct the diffusion of dissolved oxygen [36] into the microbubbles formed by ADV. The protein concentration of the fluid might also affect ADVmediated PO2 reduction by altering gas solubility [37]. The present study had three goals. The first goal was to determine the ADV pressure amplitude threshold in fluids with physiological surface tension, protein concentration, and viscosity. The second goal was to assess whether these parameters and fluid mixing affect ADV-mediated PO2 reduction. The third goal was to measure ADV-mediated PO2 reduction in whole blood, which has a higher oxygen carrying capacity (due to oxygen binding to hemoglobin in erythrocytes) than saline or plasma [38]. To achieve these goals, in vitro experiments were performed using perfluoropentane droplets diluted in fluids (i.e., phosphate-buffered

9

saline, porcine plasma, and porcine whole blood) with a physiological range of surface tensions, protein concentrations, and viscosities. Experimental set-ups with and without fluid mixing were used. Additionally, the findings obtained were compared to the predictions of the fluid PO2 at equilibrium following ADV using a previously reported numerical model [25].

2. Materials and Methods 2.1. Fluid characterization The effect of the surface tension, protein concentration, and viscosity of the fluids on ADV-mediated PO2 reduction was assessed using the following fluids: phosphate-buffered saline (0.01 M PBS, P33813, Sigma Aldrich, St. Louis, MO, USA), polyvinylpyrrolidone (PVP360, Sigma Aldrich) in PBS, and porcine plasma that was anti-coagulated with sodium citrate (Lampire Biological Laboratories, Inc., Pipersville, PA, USA). The dynamic surface tension of each fluid was measured using a twin orifice computerized surface tensiometer (SensaDyne, QC6000, CSC Scientific Company, Fairfax, VA, USA) that employed the maximum bubble pressure method [39]. Surface tension measurements were conducted for 2 min for each fluid at 37 °C. The serum protein concentration of each fluid was measured using a refractometer at room temperature, according to manufacturer instructions (ATAGO USA, Inc., Bellevue, WA, USA). Fluid dynamic shear viscosities between

10

0.5 cP and 2 cP were measured using a Size 25 calibrated Cannon-Fenske routine viscometer (Sigma Aldrich), and fluid viscosities between 3 cP and 15 cP were measured using a Size 100 calibrated Cannon-Fenske routine viscometer (Cannon Instrument Company, State College, PA, USA). The viscometers were warmed to 37 °C in a water bath, and the dynamic shear viscosity of each fluid was measured. Measurements of the surface tension, protein concentration, and viscosity of each fluid were repeated three times.

2.2. Droplet preparation Albumin-coated perfluoropentane (PFP) droplets were fabricated according to an established protocol [1]. Briefly, 0.25 mL of PFP (FluoroMed, L.P., Round Rock, TX, USA) and 0.75 mL of 4 mg/mL bovine serum albumin (BSA, A3803, Sigma Aldrich) in 0.01 M PBS (P33813, Sigma Aldrich) were added to 2 mL serum vials (Wheaton Industries Inc., Millville, NJ, USA). The vials were sealed with halobutyl rubber stoppers (Wheaton Industries Inc.), crimped, and shaken at 4,800 rpm for 30 s in an amalgamator (Wig-L-Bug, Dentsply Rinn, York, PA, USA) at 5 °C. The vials were stored at 5 °C for 24 h after high-speed shaking. Droplets were size-isolated for diameters between 1 μm to 6 μm using a twopart differential centrifugation protocol that was performed within 3 days after highspeed shaking [40]. First, droplets less than approximately 6 μm were isolated by

11

diluting 1 mL of the emulsion from each vial into 3.8 mL of PBS in a 15 mL centrifuge tube (USA Scientific, Inc. Ocala, FL, USA). The diluted emulsion was centrifuged once at 70×g for 1 min at 21 °C using an Allegra X-15R centrifuge (Beckman Coulter, Inc., Brea, CA, USA), and the supernatant (3.456 mL  0.002 mL) was reserved. Second, droplets greater than approximately 1 µm were isolated using five subsequent identical centrifugation steps. Each step consisted of centrifugation at 160×g for 1 min with the resultant supernatant (2.488 mL  0.002 mL) being discarded and the pellet being resuspended with 2.488 mL  0.002 mL of PBS. The size distribution and concentration of the droplets after size-isolation were measured using a Coulter counter (Multisizer 4, Beckman Coulter Inc., Brea, CA, USA) equipped with a 30 μm aperture. The size-isolated droplets were stored at 5 °C and used within 14 days after differential centrifugation. Because the size distribution of droplets can change over time due to Ostwald ripening [2,41], the time-dependent stability of the size-isolated droplets during storage at 5 °C was investigated. Droplet size distributions were measured 1, 4, 7, 11, 14, 21, 28, and 35 days after differential centrifugation. Five replicate measurements were averaged per sample, and three independent samples were measured at each time point. The total number density, volume-weighted concentration, number-weighted mean diameter, and volume-weighted mean diameter were computed for each time point. The total number density and volume-

12

weighted concentration at each time point were normalized to the values measured 1 day after differential centrifugation. Furthermore, the broadness of the size distribution was quantified using two different polydispersity indices (PDIs). Based on a standard for dynamic light scattering, PDI was defined as the square of the number-weighted standard deviation divided by the square of the number-weighted mean diameter [42]. Because microbubble size distributions are often non-Gaussian, several studies have defined PDIv as the ratio of the volume-weighted mean diameter to the number-weighted mean diameter [40,43,44].

2.3. Ultrasound field characterization Free-field measurements of the acoustic output and spatial beam profile of a 5 MHz single-element, focused transducer (A308S, Olympus, Waltham, MA, USA) were performed using a calibrated membrane hydrophone (0.4 mm diameter, C105040, Precision Acoustics, Dorchester, UK) mounted on a three-dimensional stepper motor-controlled system (Velmex NF90 Series, Velmex Inc., Bloomfield, NY, USA). The transducer had an aperture diameter of 1.91 cm, a focal distance of 2.54 cm, and a –6 dB ellipsoidal focal volume of 0.6 mm × 0.6 mm × 5.6 mm (azimuth × elevation × range). The transducer and hydrophone were submerged in a water tank containing degassed water. A 10-cycle sine burst was produced using an arbitrary waveform generator (AFG3500B, Keysight Technologies, Inc., Santa Rosa, CA,

13

USA), sent to a power amplifier (A300, ENI, Ltd., Rochester, NY, USA), and delivered to the transducer. After mapping the field, the acoustic output was calibrated. The focal acoustic output was measured with the hydrophone for driving voltages that produced peak negative pressures between 0 to 2 MPa (mechanical index (MI) of 0.9). Then, the peak negative pressures from 2 to 6 MPa were estimated using nonlinear numerical modeling of the acoustic field based on the Khokhlov-Zabolotskaya-Kuznetsov equation [45] in the HIFU Simulator [46]. This equation is nominally valid for transducers with an f-number greater than 1.37 [47]. The single-element transducer used in this study had an f-number of 1.33. The error associated with this smaller fnumber resulted in a 5% overestimate in peak pressures [47]. The flow phantom at the location of ultrasound insonation (section II.D) was either a 1 mm inner diameter (ID) tube (ethyl vinyl acetate (EVA), 0.38 mm wall thickness, McMaster-Carr, Aurora, OH, USA) or a 0.58 mm ID tube (polyethylene (PE), 0.19 mm wall thickness, BD IntramedicTM, Franklin Lakes, NJ, USA). Approximately 60% and 100% of the cross-sectional areas of the EVA and PE tubes were contained within the –6 dB focal volume, respectively. The acoustic attenuation through the wall of the EVA tube at 5 MHz was determined using the through-transmission, narrowband measurement technique [48] with a needle hydrophone (0.04 mm diameter, Precision Acoustics) and was measured to be 1.6

14

dB. The derated in situ peak negative pressures were computed and reported for all experiments using the EVA tube. The inner diameter of the PE tube was smaller than the housing of the needle-mounted hydrophone, precluding an in situ measurement of the acoustic attenuation coefficient. However, based on a previous study by Mazeika et al. [49], the attenuation produced by a 0.19 mm thick PE sample at 5 MHz was approximately 0.3 dB, resulting in a 3.4% difference in the pressure amplitude. Therefore, the underrated free-field peak negative pressures are reported for experiments with the PE tube.

2.4. In vitro flow phantom experiments 2.4.1. ADV pressure amplitude threshold An in vitro flow system (Fig. 1) was used to measure 1) the ADV pressure amplitude threshold [1] and 2) the PO2 in each fluid with and without ADV. To determine the ADV pressure threshold in each fluid, a flow phantom was constructed using polyvinyl chloride tubing (McMaster-Carr, Aurora, OH, USA) and EVA tubing (McMaster-Carr). The tubing was immersed in a water tank containing degassed water, maintained at 37 °C. The droplets were diluted to a concentration of 5×106 ± 0.2×106 droplets/mL in each fluid. The fluids were pumped through the flow system at 10 mL/min using a peristaltic pump (Mettler Toledo, Columbus, OH, USA). An inline thermocouple was used to confirm a temperature of 37.0 ± 0.5 °C

15

in the measurement zone. Acoustic droplet vaporization was initiated by the calibrated single-element, 5 MHz focused transducer. The transducer was aligned with the center of the EVA tubing. A 10-cycle sine burst (AFG3500B, Keysight Technologies, Inc.) was amplified (A300, ENI, Ltd.) and transmitted by the singleelement transducer. A pulse repetition period of 2 ms was used to ensure that every volume of fluid was insonified by one pulse based on a plug flow assumption. To monitor the formation of microbubbles, B-mode images of the insonified droplets were collected using an ultrasound research scanner (Vantage 256, Verasonics, Kirkland, WA, USA) equipped with a linear array transducer (L7-4, Philips, Bothell, WA, USA). The ADV pressure threshold was determined using an established protocol [50]. The in situ derated peak negative pressure of the single-element transducer was increased from 0 MPa to 4.8 MPa in steps of 0.15 MPa. Fifteen B-mode images were acquired at each pressure. A representative B-mode image of the insonified droplets in the EVA tube is shown in Figure 2A. The increased brightness outside of the tube at greater axial depths was likely a multiple-scattering artifact caused by the microbubbles. This area was not analyzed. The mean gray scale amplitude averaged across all frames was computed in a region of interest (ROI), defined within the lumen of the tube (Fig. 2A) that extended 0.9 mm across the tube lumen and 20 mm downstream of the ultrasound focus. For each sample, the mean gray scale value as

16

a function of the derated peak negative pressure was fit to a sigmoidal function. The ADV pressure amplitude threshold was defined as the midpoint of the fit [50]. A representative measurement of one sample in porcine plasma is shown in Figure 2B. The ADV threshold was measured for five separate samples for each fluid type.

2.4.2. PO2 reduction in different fluids The flow phantom included a gas exchange chamber upstream of the insonation region (Fig. 1) to adjust the partial pressure of gases in the fluid to mimic arterial gas concentrations [38]. A mixture of 14.7% O2, 5% CO2, and 80.3% N2 gases (Wright Brothers, Inc., Cincinnati, OH, USA) was humidified by bubbling the gas mixture through deionized water in a flask that served as the gas exchange chamber to achieve 100% humidity at 37 °C. The fluids containing droplets were passed through gas-permeable HelixMark® silicone tubing (0.4 mm wall thickness, Freudenberg Helix Medical, Carpinteria, CA, USA) coiled within the flask that was filled with the humidified gas mixture. To initiate ADV, the droplets were exposed to ultrasound at a 4.25 MPa peak negative pressure (MI of 1.9), which was above the ADV threshold. To investigate the time scales involved in dissolved oxygen scavenging, the PO2 in the fluid was measured at two locations that were proximal and distal to the insonation region. The PO2 was measured using a needle-type optical sensor (OXR430-UHS,

17

Pyroscience, Aachen, Germany) inserted into the flow phantom with a hemostasis valve and a flow-through optical sensor (OXFTC, Pyroscience). Both sensors measured PO2 using the same optical technology. In preliminary experiments without ADV (0 MPa insonation), the PO2 measurements of a sensor located approximately 9 cm upstream of the insonation region were the same as those obtained

approximately

9

cm

downstream.

Therefore,

subsequent

PO2

measurements were only obtained at downstream locations. The needle and flowthrough sensors were located either at 4.7 cm and 16.7 cm, respectively, or 9.5 cm and 26.5 cm, respectively, downstream of the insonation region depending on the flow rate, as described later (Fig. 1). The distances were selected such that the emulsion took 2 s and 10 s to travel from the insonation region to the needle-type sensor (i.e., PO2 sensor 1) and the flow-through sensor (i.e., PO2 sensor 2), respectively. The relatively small size and geometry of the needle-type sensor allowed placement closer to the insonation region than the flow-through sensor. The flow-through sensor had a larger geometric footprint and was placed distal to the insonation region. The PO2 was averaged over 20 s measurement periods corresponding to times without ADV and with ADV (4.25 MPa insonation, MI of 1.9) for each sample. The PO2 reduction for each sensor was computed as the difference in the measurements with and without ADV. The two measured PO2

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reductions were averaged and defined as ΔPO2. Five samples were investigated for each fluid. The effluent from the flow system was collected (Fig. 1), and the size distributions of the droplets in the effluent were measured using the Coulter counter. The PO2 at equilibrium was predicted using a previously reported numerical model [25] that employed size distribution measurements as inputs. These results were compared with the experimental PO2 measurements. The following parameters were incorporated in the numerical model: arterial gas partial pressures (i.e., PN2 of 568 mmHg, PO2 of 105 mmHg, PCO2 of 40 mmHg, and vapor pressure of water of 47 mmHg) [38], an ambient hydrostatic pressure of 1 atm, and a temperature of 37 °C. Perfluoropentane was assumed to be insoluble in all fluids. Oxygen solubility was assumed to be 964 L atm/mol in water [51], 1014 L atm/mol in saline [51], and 1047 L atm/mol in human plasma [52,53]. A priori simulations showed that varying the solubility parameter over the range of 964 to 1047 L atm/mol yielded PO2 values that were different by at most 2.5 mmHg. Therefore, the oxygen solubility of water was used for all fluids to simplify the calculation. The numerical model did not incorporate effects due to surface tension, viscosity, and protein concentration. Three tubing configurations at the location of and distal to the insonation region of the flow system were used (Figs. 1 and 3). The first tubing configuration consisted of the 0.58 mm ID PE tube, in which the –6 dB focal zone of the single-

19

element transducer encompassed the entire tube cross-section (Fig 3A). The pulse repetition period was 1.3 ms, and the flow rate was 5 mL/min, to ensure that each volume of fluid was exposed to one ultrasound pulse under a plug flow assumption. The second tubing configuration consisted of the 1 mm ID EVA tube, similar to the diameter of a rabbit coronary artery [54]. A flow rate of 10 mL/min and pulse repetition period of 2 ms were employed to insonify all droplets once under the plug flow assumption. In the third tubing configuration, a fluid mixing chamber was added 3 cm distal to the insonation region (1 mm ID tube). Two bifurcating and intersecting 1.6 mm ID tubes were used to promote fluid mixing distal to the –6 dB ultrasound focal volume (Fig. 3C). The PO2 in porcine whole blood was measured using the in vitro flow phantom (Fig. 1) with the 0.58 mm ID tube (Fig. 3A). Porcine whole blood that was anti-coagulated with citrate phosphate dextrose (Lampire Biological Laboratories, Inc.) was used for ADV experiments. The PO2 in whole blood was measured without ADV (0 MPa) and with ADV (4.25 MPa). The oxygen saturation of hemoglobin (SaO2) in the porcine whole blood was calculated using the measured PO2 and the following equation reported by Serianni et al. [55]: SaO2 (%) = (0.13534 × PO2)3.02 / [(0.13534 × PO2)3.02 + 91.2] × 100. (1)

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2.5. Statistical analyses The normality of all replicate measurements for each experimental arm was confirmed using the Kolmogorov-Smirnov test. The ADV pressure threshold measurements in the different fluids were compared using a one-way analysis of variance (ANOVA). Two-way ANOVA was used to determine whether the fluid property and sensor position affected the PO2 measurements. The ΔPO2 values were compared among the different fluids using one-way ANOVA. All statistical tests were performed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). A p-value of less than 0.05 was used to denote significant differences for all statistical tests.

3. Results 3.1. Fluid characterization Measurements of the surface tension, protein concentration, and viscosity of each fluid are presented in Table 1. The viscosity of the 3 mg/mL PVP solution was similar to that of porcine plasma, but the 3 mg/mL PVP did not contain protein. Whole blood viscosity depends on the hematocrit level and shear rate of the blood in the measurement apparatus [28]. At a physiological hematocrit of 45% and arterial shear rate greater than 40 s-1, viscosity was approximately 4 cP at 37 °C [28]. The 15 mg/mL PVP solution had a viscosity similar to that of whole blood at 37 °C

21

[28,56]. The surface tension in PBS was higher than those of the other fluid types, whose measured surface tension values differed within approximately 3 mN/m of each other.

3.2. Droplet characterization The number-weighted and volume-weighted size distributions of the droplets are shown in Figures 4(A, B). The peak number and peak volume decreased as a function of storage time at 5 °C, and the size distributions shifted to larger droplet diameters. The total number density and volume-weighted concentration as a function of storage time are shown in Figure 4C. The polydispersity indices are shown in Figure 4D. Based on these results, the size-isolated droplets were used within 14 days after differential centrifugation for all subsequent experiments. Because the number density of the droplets decreased with time (Fig. 4C), the density of droplets in the stored stock emulsion was determined prior to each experiment using the Coulter counter. Subsequently, the droplets were diluted to obtain a total number density of 5×106 ± 0.2×106 droplets/mL in each fluid for ADV experiments.

3.3. ADV pressure amplitude threshold

22

The ADV pressure threshold measurements for each fluid are shown in Figure 5. The ADV threshold measurements among the various fluids were not statistically different (p = 0.87). These results indicate that in the physiologic range tested, surface tension, protein concentration, and viscosity of the fluids (within the ranges tested) did not affect the ADV threshold.

3.4. PO2 reduction in fluids with physiological surface tension, protein concentration, and viscosity For all experiments without ADV (i.e., 0 MPa insonation) using the three tubing configurations, the differences in the measured PO2 between sensors 1 and 2 were not statistically significant (p > 0.05). Additionally, the measured PO2 without ADV was significantly higher than those with ADV for all experiments (p < 0.0001). The PO2 measurements for each fluid using the 0.58 mm ID tube are presented in Supplementary Figure S1. The difference between the PO2 measured with sensors 1 and 2 during ADV were not significant (p = 0.37). The average PO2 reduction (ΔPO2) corresponding to both sensors are shown in Table 1 for each fluid. No consistent trends were observed between the ΔPO2 values and the fluid properties (Table 1). Additionally, the difference between the ΔPO2 values for each fluid and the model prediction at equilibrium was within 17% (Fig. 6).

23

Two-way ANOVA was performed to assess for differences in PO2 measurements between sensors and between fluid types. The PO2 measurements for each fluid using the 1 mm ID tube with and without mixing are shown in Supplementary Figure S2. A difference in PO2 measurements between sensors was observed (p < 0.05). Similarly, a difference was observed between fluid types without mixing (p < 0.05), but not with mixing (p = 0.15). The difference in the PO2 measurements between sensors increased as a function of fluid viscosity without mixing (Fig. 7A). However, for experiments both with and without mixing, no consistent trend was observed with respect to surface tension and protein concentration. Without mixing, a significant difference in ΔPO2 was observed with respect to fluid type using a one-way ANOVA (p < 0.05). The ΔPO2 decreased with increasing fluid viscosity (Fig. 7B), but no consistent trends were observed with respect to fluid surface tension and protein concentration. With mixing, the ΔPO2 values were not significantly different (p = 0.47).

3.5. PO2 reduction in whole blood The measured PO2 and calculated SaO2 in whole blood with and without ADV are shown in Figure 8. Without ADV, the measured PO2 was 110 ± 2 mmHg (Fig. 8A), corresponding to a calculated SaO2 of 97.5% ± 0.1% (Fig. 8B). With ADV,

24

sensor 1 measured a PO2 of 94 ± 3 mmHg (Fig. 8A), corresponding to a PO2 reduction of 16.1 ± 3.4 mmHg and a calculated SaO2 of 96.0% ± 0.4% (Fig. 8B). Sensor 2 measured a PO2 of 81 ± 2 mmHg (Fig. 8A), corresponding to a PO2 reduction of 30.8 ± 3.2 mmHg and a calculated SaO2 of 93.8% ± 0.5% (Fig. 8B). The measured PO2 and calculated SaO2 decreased significantly from sensors 1 to 2 with ADV (p < 0.05).

4. Discussion 4.1. Fluid characterization In the current study, surface tension measurements of each fluid were found to be similar to those reported for normal human serum [57]. The duration of the surface tension measurement (2 min) was comparable to that of a single ADV experiment. However, this duration was significantly longer than the time needed for the fluid to flow from the ultrasound insonation region to either of the dissolved oxygen sensors. Thus, the dynamic surface tension [58] might have been different from the measured surface tension. Additionally, the microbubbles produced by ADV were likely coated irregularly with the droplet surfactant, bovine serum albumin [24], which may also have modified the interfacial surface tension. In the present study, the measured protein concentration in porcine plasma was within the range for healthy humans [59] and pigs [60]. Note that the

25

refractometer measurements were performed at room temperature, as per instructions provided by the manufacturer. These measurements were not conducted at 37 °C because varying the temperature of the sample fluid could affect refractometer readings due to changes in the refractive index [61]. The viscometers used in this study were designed for Newtonian liquids at shear rates between 153 and 2830 s-1 (Cannon Instrument Company). Blood plasma and PVP solutions exhibit Newtonian behavior within this range of shear rates [62,63]. In the present study, the measured dynamic viscosity of porcine plasma was similar to that reported previously for humans [64,65]. The 15 mg/mL PVP solution had the same viscosity as whole blood, which was reported previously at a physiological hematocrit of 45% and arterial shear rate greater than 40 s-1 [28].

4.2. Droplet characterization Differential centrifugation [43,66] was used in this study to isolate perfluoropentane droplets between 1 to 6 μm in diameter. Droplets with diameters less than 1 μm were removed and relatively few of these droplets transitioned phase during ultrasound exposure [43,66]. Droplets with diameters greater than 6 μm were removed to limit the potential occurrence of gas emboli in future in vivo studies. Before ingassing, the largest droplets used in this study (approximately 6 m in diameter) would be expected to produce 31.8 μm microbubbles based on the radial

26

expansion factor associated with the phase transition [1]. The likelihood of a microbubble to embolize a vessel is complex and depends not only on the microbubble and vessel sizes, but also on the microbubble shell properties, the local blood pressure, and the endothelial lining of the vessel [67]. Butler and Hills [68] reported that virtually all injected microbubbles greater than 22 m in diameter were filtered out of circulation in canine lungs. Based on the size distributions reported in Figure 4A, approximately 9% of the droplets used in this study would form microbubbles greater than 22 m in diameter. The mean droplet diameter of the number-weighted and volume-weighted size distributions increased over storage time (Figs. 4A,B). Additionally, the number density of droplets decreased while the total volume-weighted concentration remained constant. These changes were consistent qualitatively with Ostwald ripening [69]. However, the polydispersity of the size distribution increased over time, which was inconsistent with classical theories of Ostwald ripening [69]. These observations indicate that the temporal evolution of the droplets during storage requires further investigation.

4.3. ADV pressure threshold In a series of in vitro flow phantom experiments, we found that the ADV pressure threshold for droplets with diameters between 1 and 6 m was not affected

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by the surface tension, protein concentration, or viscosity of the fluid (Fig. 5). Previously, Fabiilli et al. [29] investigated the effects of fluid viscosity and surface tension on the ADV threshold of perfluoropentane droplets exposed to 3.5 MHz ultrasound. Their droplets ranged in size from sub-micron to greater than 10 m. They reported that the surface tension of the fluid did not affect the ADV threshold [29], which is consistent with the experimental findings of the present study (Fig. 5). Contrary to our observations, Fabiilli et al. [29] found that an increase in the fluid viscosity led to an increase in the ADV threshold. The differences in the droplet size range and the ultrasound frequency used could explain this discrepancy. In particular, the droplets greater than 10 μm used by Fabiilli et al. [29] might have transitioned at lower pressure amplitudes and with the greatest efficiency (compared to the droplet sizes in our study), which would have impacted their measured ADV thresholds [18,25,29,40,70,71].

4.4. PO2 reduction in blood mimicking fluids and plasma No consistent trend in the ΔPO2 values was observed with respect to fluid surface tension (Table 1, Fig. 6). The lack of a trend may be due to the relatively small change in surface tension values investigated in this study (Table 1). Indeed, there was only a 9% difference between the highest and lowest fluid surface tension measured. A larger range of surface tension values was not investigated because this

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study focused on a physiologically relevant range. If smaller droplets were used (e.g., less than 1 μm in diameter), such as those produced by the condensation manufacturing approach [41], the extent of PO2 reduction in relation to surface tension may have been greater. The concentration of porcine albumin in plasma may impact oxygen scavenging in two ways. First, dissolved molecular porcine albumin could obstruct oxygen diffusion into the microbubbles [36]. Second, the dissolved porcine albumin might adsorb onto patches of free gas-fluid interfaces on the microbubbles, not covered by the BSA coating [24], thereby modifying the diffusion constant of oxygen into the microbubble or by changing the interfacial surface tension [32]. Nonetheless, our results did not show an effect consistent with either of these mechanisms (Table 1, Fig. 6). Dissolved albumin did not appear to have a substantial effect on the diffusion coefficient of dissolved oxygen through the fluid. This observation may be, in part, explained by the fact that protein adsorption typically occurs on a time scale of tens of minutes [72], while ingassing occurs on a time scale of milliseconds to seconds [1,23,73]. Fluid viscosity was the only fluid parameter observed to affect the measured PO2 (Fig. 7). The effect was observed when the ultrasound focal region was smaller than the tube cross-section and in the absence of mixing (Figs. 3B and 7). The differences in the PO2 measurements between the sensors under these conditions

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may have been due to the rate of ingassing varying with viscosity, which may occur in two ways. First, the diffusion coefficient decreases with increasing dynamic shear viscosity [31]. A slower diffusion rate would increase the time needed for oxygen to diffuse through the fluid and into the microbubbles. Second, less mixing occurs in fluids with higher viscosities due to a lower Reynolds number. Reduced mixing between fluid regions with and without transitioned PFP microbubbles (Fig. 3B) could result in larger diffusion path lengths between oxygen molecules and microbubbles. Correspondingly, the time required for the PO2 in the fluid to reach equilibrium would increase. Fluid mixing reduces diffusion path lengths, but does not affect the diffusion coefficient [31]. This concept is consistent with our finding that the addition of fluid mixing channels downstream of the insonation region (Fig. 3C) resulted in similar PO2 measurements regardless of the fluid viscosity (Fig. 7). Therefore, these results suggest that viscosity affects PO2 due to the absence of mixing. The effects of viscosity and diffusion rates could also be elucidated in the future by studying the kinetics of ADV-induced PO2 changes. Additionally, it was observed in two cases with mixing (PBS and 15 mg/mL PVP), that the proximal sensor measured a lower PO2 than the distal sensor (Supplementary Fig. S2). This result does not correlate with any of the measured fluid parameters. The results in Figs. 6 and 7B showed that ΔPO2 values were greater in the 0.58 mm ID tube than in the 1 mm ID tube. Higher ΔPO2 values in the 0.58 mm ID

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tube may be due to the -6 dB focal region covering a larger fraction of the lumen. Note that it is possible that the pressure amplitudes in the two tubes were not the same. Although the pressure amplitude was assessed in the 1 mm ID tube, the 0.58 mm ID tube did not allow pressure amplitude calibration in situ. In addition, estimation of the in situ pressure amplitude in the 0.58 mm ID tube could have been confounded by reverberation within the lumen [74]. It is difficult to determine the effects of these factors on the ADV transition efficiency and the ΔPO2. The numerical model did not incorporate surface tension, protein concentration, and viscosity of the fluid. However, the predicted ΔPO2 was within 17% of the measurements when the ultrasound focal region encompassed the entire tube cross-section (Fig. 6). This outcome provides additional evidence that surface tension, protein concentration, and viscosity (in the physiological range) play a relatively minor role in PO2 reduction.

4.5. PO2 reduction in whole blood The PO2 reduction in whole blood was less than that in the 15 mg/mL PVP solution (Table 1), which was likely due to the presence of hemoglobin in erythrocytes. Note that in whole blood, approximately 2% of the oxygen is dissolved in the plasma and 98% is bound to hemoglobin [38]. When the PO2 decreases, oxygen unbinds from hemoglobin [38], thereby attenuating the PO2 reduction. The

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calculated SaO2 decreased by approximately 3.7% with ADV, based on the PO2 measured by sensor 2 (Figure 8). This decrease corresponded to scavenging 384 µM of O2 from whole blood – approximately 344 µM and 40 µM were scavenged from hemoglobin and plasma, respectively. By comparison, approximately 65 µM of O2 were scavenged from the 15 mg/mL PVP solution. Taken together, six times more O2 molecules were scavenged in whole blood than in 15 mg/mL PVP. However, in whole blood, nearly nine times more O2 was scavenged from the erythrocytes than from the plasma. Additionally, the PO2 may not have reached equilibrium by the time the whole blood arrived at sensor 2 (Fig. 8A). The PO2 measured by sensors 1 and 2 were significantly different for whole blood, unlike for the other fluids. These results could be due to 1) the diffusion coefficient of oxygen in whole blood and 2) the hemoglobin-oxygen dissociation kinetics. The diffusion coefficient of oxygen through plasma decreases in the presence of erythrocytes [75]. Furthermore, oxygen association and dissociation from hemoglobin occurs on the order of deciseconds to seconds [38,76,77]), which is similar to the duration of our experimental measurements. To quantify dissolved oxygen scavenging in whole blood during ADV, the SaO2 should be measured simultaneously with the PO2 to compensate for this limitation.

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4.6. Potential for bioeffects in vivo In vivo studies are necessary to elucidate the potential for beneficial or deleterious bioeffects due to ADV-mediated oxygen scavenging. In the present study, the measured PO2 and calculated SaO2 in whole blood with ADV (Fig. 8) were within the human arterial blood-gas range [38]. Based on the numerical model by Radhakrishnan et al. [25], increasing the amount of perfluoropentane gas via ADV would lower the PO2. At a constant droplet concentration, increasing the fraction of droplets converting to microbubbles would thus lower the PO2. In the current study, a higher fraction of phase-transitioned droplets, and consequently, a higher PO2 reduction, were achieved in a tube with an inner diameter smaller than the ultrasound insonation beamwidth (Figs. 3A and 6, Table 1). For in vivo studies, judicious selection of the ultrasound transducer would be important for insonifying vessels of interest with specific diameters. Furthermore, ultrasound insonation parameters can be modified to increase the phase-transition efficiency. Additionally, Fabiilli et al. [29] and Mercado et al. [40] demonstrated that as the droplet diameter increased, the fraction of phase-transitioned droplets increased. Therefore, droplets larger than those employed in the current study (> 6 m) could be used to enhance PO2 reduction. However, droplets larger than 6 m could increase the likelihood of embolization by microbubbles formed after ADV [10,78]. Another approach to

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modulating the amount of perfluoropentane gas would be to modify the droplet concentration.

5. Conclusions The surface tension and protein concentration in the fluid, within the ranges tested, had a negligible effect on ADV-mediated PO2 reduction. Fluid viscosity was demonstrated to have a moderate transient effect on PO2 reduction when the ultrasound focus was smaller than the fluid volume. Fluid mixing accelerated PO2 reduction regardless of the viscosity. The PO2 reduction in whole blood (30.8 ± 3.2 mmHg) was demonstrated to be less than that in a PVP solution (40.2 ± 2.1 mmHg) with the same viscosity, suggesting complex dynamics in the presence of erythrocytes. These findings can help guide the use of ADV for diagnostic imaging and therapy.

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Acknowledgements The authors would like to thank Kenneth Bader, Ph.D., for assistance with the viscometers and the HIFU simulator used in this study. This work was supported, in part, by the National Heart, Lung, and Blood Institute [grant number K25 HL133452]; the National Institute of Neurological Disorders and Stroke [grant number R01 NS047603]; and the American Heart Association [grant number 16SDG27250231].

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Figure captions

Figure 1. Schematic of the in vitro flow system. Droplets were pumped from the sample reservoir through the flow system maintained at 37 °C. The fluid was gassaturated to arterial blood partial pressures in a gas exchange chamber and exposed to 5 MHz pulsed ultrasound using the single-element transducer. A Verasonics Vantage 256 scanner, equipped with an L7-4 linear array, was used to monitor the formation of microbubbles during ADV. The PO2 in the fluid was measured by two sensors located downstream of the insonation region. The effluent was collected to measure the size distribution of droplets that did not undergo ADV. (Color online)

Figure 2. (A) A representative B-mode image of acoustic droplet vaporization (ADV) in a flow phantom. The blue dashed box denotes the region in which the mean gray scale value was computed. (B) The mean gray scale value within a region of interest in the B-mode image was plotted as a function of the peak negative pressure in situ. Measurements for a representative droplet sample in porcine plasma are shown. The experimental data was fit to a sigmoidal function. The ADV threshold was defined as the peak negative pressure at the midpoint of the sigmoidal function. A.U. = arbitrary units. (Color online)

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Figure 3. Tubing configurations. (A) A schematic of the cross-section of a 0.58 mm inner diameter (ID) tube, in which the entire fluid volume was exposed to ultrasound in the ellipsoidal –6 dB focal region. Several droplets (blue circles) phasetransitioned into microbubbles (yellow circles) that scavenged dissolved oxygen (green O2) from the surrounding fluid. (B) A schematic of the cross-section of a 1 mm ID tube in which the focal region did not encompass the entire tube crosssection. (C) Fluid mixing channels, composed of bifurcating and intersecting tubes and y-connectors, were added downstream of the insonation region in the 1 mm ID tube and upstream of the PO2 sensors. (Color online)

Figure 4. Stability of droplets during 35 days of storage in 5 °C. (A) Numberweighted and (B) volume-weighted size distributions of droplets measured at 1, 4, 7, 11, 14, 21, 28, and 35 days of storage. (C) The total number density (squares) and total volume-weighted concentration (triangles) at each time point, relative to the total values measured after 1 day of storage. (D) The polydispersity indices, PDIv and PDIσ, as a function of storage time. The mean and standard deviation of measurements for three samples are shown for each time point. (Color online)

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Figure 5. Measurements of ADV pressure threshold for each fluid used in the study. The height of the bars represents the mean ADV threshold measurements for five samples. The error bars denote the standard deviation of the measurements.

Figure 6. The measured and predicted PO2 reduction (ΔPO2) as a function of the volume of perfluoropentane (PFP) phase-transitioned in the 0.58 mm inner diameter tube. The PFP volume is reported as a percent of the total fluid volume. The mean and standard deviation of measurements in five samples of each fluid are shown. (Color online)

Figure 7. (A) The difference between the PO2 measured by sensor 1 (P1) and sensor 2 (P2) and (B) the average PO2 reduction (ΔPO2) across both sensors as a function of fluid viscosity with and without mixing in the 1 mm inner diameter tube. The mean and standard deviation of measurements in five samples of each fluid are shown. (Color online)

Figure 8. (A) The measured PO2 and (B) the calculated SaO2 in porcine whole blood with and without ADV. The mean and standard deviations of PO2 and SaO2 values for five samples are shown. Significant differences (p < 0.05) in PO2 and SaO2 values are denoted by the asterisks.

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Tables Table 1. Measured dynamic surface tension, protein concentration, and dynamic shear viscosity of the different fluids. The mean and standard deviation of three measurements of each fluid are reported. The uncertainty for the viscosity and surface tension were determined by inter-sample variability. The uncertainty for the protein concentration was determined by the refractometer’s resolution. The mean and standard deviation of the measured PO2 reductions, ΔPO2, are shown for five samples of each fluid in the 0.58 mm inner diameter tube.

Fluid type

Dynamic Protein surface tension concentration (mN/m) (mg/mL)

Dynamic shear viscosity (cP)

(mmHg)

ΔPO2

0.01 M PBS

68.1 ± 0.5

0.0 ± 0.0

0.74 ± 0.01

35.7 ± 2.7

Porcine plasma

62.2 ± 1.1

68.7 ± 0.0

1.20 ± 0.01

42.8 ± 3.5

3 mg/mL PVP

64.7 ± 0.2

0.0 ± 0.0

1.18 ± 0.02

44.2 ± 2.4

15 mg/mL PVP

65.1 ± 0.1

0.0 ± 0.0

4.04 ± 0.05

40.2 ± 2.1

Tables

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Table 1. Measured dynamic surface tension, protein concentration, and dynamic shear viscosity of the different fluids. The mean and standard deviation of three measurements of each fluid are reported. The uncertainty for the viscosity and surface tension were determined by inter-sample variability. The uncertainty for the protein concentration was determined by the refractometer’s resolution. The mean and standard deviation of the measured PO2 reductions, ΔPO2, are shown for five samples of each fluid in the 0.58 mm inner diameter tube.

Fluid type

Dynamic Protein surface tension concentration (mN/m) (mg/mL)

Dynamic shear viscosity (cP)

(mmHg)

ΔPO2

0.01 M PBS

68.1 ± 0.5

0.0 ± 0.0

0.74 ± 0.01

35.7 ± 2.7

Porcine plasma

62.2 ± 1.1

68.7 ± 0.0

1.20 ± 0.01

42.8 ± 3.5

3 mg/mL PVP

64.7 ± 0.2

0.0 ± 0.0

1.18 ± 0.02

44.2 ± 2.4

15 mg/mL PVP

65.1 ± 0.1

0.0 ± 0.0

4.04 ± 0.05

40.2 ± 2.1

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CRediT Author Statement

Karla P. Mercado-Shekhar: Conceptualization, Methodology, Software, Formal Analysis, Validation, Investigation, Writing – Original Draft, Visualization. Haili Su: Investigation. Deepak S. Kalaikadal: Investigation. John N. Lorenz: Methodology, Resources, Writing – Original Draft, Supervision. Raj M. Manglik: Methodology, Resources, Writing – Original Draft. Christy K. Holland: Conceptualization, Methodology, Resources, Writing – Original Draft, Supervision. Andrew N. Redington: Conceptualization, Writing – Original Draft, Supervision. Kevin J. Haworth: Conceptualization, Methodology, Formal Analysis, Resources, Writing – Original Draft, Supervision, Project Administration, Funding Acquisition.

Highlights:  Physiological surface tension had a negligible effect on ADV-mediated PO2 reduction.  Fluid protein concentration was uncorrelated with PO2 reduction.

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 Viscosity had a transient effect on PO2 reduction.  Fluid mixing accelerated PO2 reduction at physiologic viscosities.  Reduction in the PO2 in whole blood was demonstrated.

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