Role of Surface Tension in Gas Nanobubble Stability Under Ultrasound

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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 9949−9956

Role of Surface Tension in Gas Nanobubble Stability Under Ultrasound Christopher Hernandez,†,∥ Lenitza Nieves,‡,∥ Al C. de Leon,‡ Rigoberto Advincula,§ and Agata A. Exner*,‡,† †

Department of Biomedical Engineering, ‡Department of Radiology, and §Department of Macromolecular Science and Engineering, Case Western Reserve University, Cleveland, Ohio 44106, United States ABSTRACT: Shell-stabilized gas nanobubbles have recently captured the interest of the research community for their potential application as ultrasound contrast agents for molecular imaging and therapy of cancer. However, the very existence of submicron gas bubbles (especially uncoated bubbles) has been a subject of controversy in part due to their predicted Laplace overpressure reaching several atmospheres, making them supposedly thermodynamically unstable. In addition, the backscatter resulting from ultrasound interactions with nanoparticles is not predicted to be readily detectable at clinically relevant frequencies. Despite this, a number of recent reports have successfully investigated the presence and applications of nanobubbles for ultrasound imaging. The mechanism behind these observations remains unclear but is thought to be, in part, influenced heavily by the biophysical properties of the bubblestabilizing shell. In this study, we investigated the effects of incorporating the triblock copolymer surfactant, Pluronic, into the lipid monolayer of nanobubbles. The impact of shell composition on membrane equilibrium surface tension was investigated using optical tensiometry, using both pendant drop and rising drop principles. However, these techniques proved to be insufficient in explaining the observed behavior and stability of nanobubbles under ultrasound. Additionally, we sought to investigate changes in membrane surface tension (surface pressure) at different degrees of compression (analogous to the bubble oscillations in the ultrasound field) via Langmuir−Blodgett experiments. Results from this study show a significant decrease (p < 0.0001) in the nanobubble equilibrium surface tension through the incorporation of Pluronic L10, especially at a ratio of 0.2, where this value decreased by 28%. However, this reduction in surface pressure was seen only for specific compositions and varied with monolayer structure (crystalline phase or liquid-crystalline packing). These results indicate a potential for optimization wherein surface pressure can be maximized for both contraction and expansion phases with the proper lipid to Pluronic balance and microstructure. KEYWORDS: ultrasound contrast agents, microbubbles, nanobubbles, surface tension, Langmuir−Blodgett, pendant drop

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less than 200 nm.7 However, this effect is highly heterogeneous and has been found to depend on many factors, including tumor type, nanoparticle shape8 and particle mechanical properties.9 Studies have shown bacteria as large as 1000 nm10 to accumulate in tumors. Although submicron gas bubbles, commonly referred to as bulk nanobubbles or ultrafine bubbles (ISO/TC281), could be revolutionary in ultrasound imaging, their persistence in aqueous solutions was initially a subject of significant controversy.11 This is in part due to the size-dependent stability of an uncoated nanobubble that is predicted by the Laplace pressure. As shown in eq 1, this pressure (P) is dependent on the bubble radius (R) and the surface tension (σ).

INTRODUCTION Clinically available ultrasound contrast agents (UCAs) are micron-sized gas bubbles that are stabilized by either lipids or proteins and act as ultrasound echo enhancers. Although their approval and primary indications vary from country to country, their clinical applications are typically focused on quantifying tissue perfusion, microvascular blood flow, and blood volume to improve the quality of echocardiograms and the detection of cancerous focal liver lesions.1 This successful clinical acceptance and use of UCAs have been, in part, due to the wide availability, low cost and outstanding safety profile of ultrasound imaging. Although the large, 1−10 μm, microbubble size has been ideal for intravascular applications, this larger footprint narrows the scope of potential applications, especially in cancer molecular imaging and theranostics.2−4 To surmount vascular confinement and develop agents for cancer detection, there has been a recent interest in the development of submicron bubble populations. As is widely accepted, tumors typically possess defective vascular architecture, which allows for the passive movement of nanoscale vehicles outside of the vasculature and accumulation within the tumor parenchyma via the enhanced permeability and retention effect.5,6 Maximal accumulation is typically seen for particles © 2018 American Chemical Society

ΔP = Pinside − Poutside =

2σ R

(1)

As the radius decreases to the nanometer range, this pressure difference increases to several atmospheres, making the Received: December 28, 2017 Accepted: March 1, 2018 Published: March 1, 2018 9949

DOI: 10.1021/acsami.7b19755 ACS Appl. Mater. Interfaces 2018, 10, 9949−9956

Research Article

ACS Applied Materials & Interfaces

Figure 1. Illustration demonstrating the fabrication process for Pluronic nanobubbles.

nanobubbles thermodynamically unstable. Additionally, bubble stability is related to the counteractive forces of the partial pressures of dissolved gases in the surrounding fluid. A more comprehensive predictive model for bubble stability, proposed by Epstein and Plesset in 1950, predicts that an uncoated submicron bubble, in a gas-saturated medium would have a lifetime of less than 1 s.12 Contrary to what these two equations predict, naturally occurring oceanic nanobubbles have been observed to be stable for over 20 h.13 These bubbles were proven to be coated with naturally occurring surfactants that reduced their surface tension. Evidence of a compressible gas core was demonstrated by applying positive and negative pressures, in which the bubbles were shown to expand and contract in size.13 To study the mechanisms behind their stability, the majority of nanobubble research has been done on surface nanobubbles. Unlike bulk nanobubbles, which are spherical and completely surrounded by a liquid medium, surface nanobubbles are gasfilled pockets on a surface with a spherical cap (for recent reviews see refs 14−16). Surface nanobubbles were initially discovered and imaged using atomic force microscopy, and their existence was later confirmed with other techniques.15 Various theories, including contamination of their surface,17 the dynamic equilibrium theory,18 and pinning,19 have been suggested as a mechanism for their anomalous stability. Building upon these findings, a number of recent reports have examined various shell-stabilized nanoparticle formulations for submicron contrast agents for their use in ultrasoundguided cancer detection and theranostics.20,21 These formulations have ranged from echogenic liposomes22,23 and phaseshifting nanoemulsions24 to polymeric or lipid-coated nanobubbles.25−28 These particles act as echo enhancers due to high acoustic impedance difference between the gas and blood. Although they have shown promise in preclinical studies, for many of them the critical stabilization strategy is a stiff membrane, which results in high resonant frequencies. With higher resonant frequencies, there is an increased mismatch with clinically relevant ultrasound transducer frequencies,29

which ultimately harm image quality. Because the viscoelastic properties of the stabilizing shell are known to have a large impact on microbubble activity within the ultrasound field, much effort has been dedicated to gaining an in-depth understanding of these processes.30−33 Building upon this foundation, our group has previously reported on formulation of nanobubbles stabilized by a combination of phospholipids and a synthetic polymer surfactant, called Pluronic.34−37 Pluronic is a nonionic triblock copolymer composed of repeating subunits of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), following the general structure of PEOx−PPOy−PEOx. Various Pluronics have been shown to intercalate in lipid monolayers and reduce the surface tension at the air−water interface by folding into a V-shape (Figure 1), with the center PPO chains interacting with the hydrophobic lipid acyl chains and the outer PPO segments interacting with the hydrophilic lipid polar head groups.38−40 Evidence of a gas core in these constructs has been demonstrated with cryo-EM41 and using the resonant mass measurement technique.42 Additionally, these bubbles have strong ultrasound contrast enhancement, which is consistent with a compressible gas core as opposed to oil droplets.42,43 Although the specific mechanisms remain unclear, the nanobubbles we fabricated have been shown to persist stably both in vitro and in vivo and have been visualized with standard clinical ultrasound equipment. It is also of interest to further increase their stability, as in vivo circulation time is critical for their use as cancer detection agents. We hypothesize that the incorporation of Pluronic into the formulation stabilizes nanobubbles by imparting additional barrier robustness to counteract applied pressure as a consequence of the reduction in its membrane’s surface tension. Numerical simulations of nanobubble nonlinear dynamics have also shown that a significant decrease in surface tension with the addition of Pluronic could be responsible for their unexpected signal at clinical ultrasound frequencies.44 In the current report, we investigated the effects of varying Pluronic−lipid ratios in the bubble shell on nanobubble behavior in vitro. Specifically, the 9950


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two-dimensional gain of 70 dB, imaged for 15 min. Images were processed using the onboard CHI-Q quantification software. Regions of interest (ROI) with the same dimensions and depth was drawn for each study. The mean signal intensity per ROI for each image was acquired, and the time−intensity curves were fitted. Signal decay was assumed to be a first-order reaction. Experiments were carried out in triplicate. Langmuir−Blodgett Trough. To evaluate the effect of Pluronic L10 on the surface tension of the lipid monolayer, Langmuir−Blodgett technique was used. In these experiments, surface pressure−area isotherms were obtained using a KSV Instruments Teflon Langmuir trough equipped with two identical mobile barriers and a Wilhelmy plate (KSV Instruments). Ultrapure water (resistivity ∼ 18 MΩ·cm) was used as the subphase. Lipid−surfactant solutions were made by dissolving the DPPC, DPPA, DPPE, and mPEG-DSPE in a mass ratio of 4:1.5:1:1 (1 mg/mL final concentration) and varying amounts of Pluronic L10 in 1 mL of chloroform. Final Pluronic L10:lipids molar ratios ranging from 0.002 to 0.6 were achieved. A total of 60 μL of the lipid−surfactant solution was spread throughout the trough. The trough equilibrated for 10 min to allow for chloroform evaporation. Compression rate was set at 10 mm/min. Control solutions were made by dissolving the lipid mixture in 1 mL of chloroform without the addition of Pluronic L10. Experiments were conducted at room temperature and repeated in triplicate. Statistics. Data were presented as mean value ± standard deviation. Two-tailed, unpaired Student’s t test was used for comparisons of all groups. A p value < 0.05 was considered significant. Grubb’s test was used to determine outliers in the groups.

impact of shell composition on membrane equilibrium surface tension was investigated using optical tensiometry, using both pendant drop and rising drop principles. Kinetic or quasiequilibrium surface tension, which better describes bubble stability under an ultrasound field was investigated via Langmuir−Blodgett analysis.

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EXPERIMENTAL SECTION

Materials. The lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) were obtained from Corden Pharma (Switzerland), and 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (mPEG-DSPE) (ammonium salt) was obtained from Laysan Lipids (Arab, AL). Pluronic L10 was donated by BASF (Shreveport, LA). All materials were used as received. Surface Tension Using the Pendant Drop Principle. Lipid films were prepared by dissolving the lipids in chloroform followed by the evaporation of the solvent using a hot plate at 75 °C. Films were then hydrated in 1 mL of a L10 solution in phosphate-buffered saline (PBS) (pH 7.4) at varying Pluronic L10:lipids molar ratios, ranging from 0.002 to 0.6, on a water bath at 75 °C for 30 min. Upon cooling the solutions to room temperature, a drop of the solution was dispensed on the tip of a Hamilton syringe equipped with a needle with an outer diameter of 0.7 mm. Images of three replicate drops were acquired using a KSV CAM 200 Optical Contact Angle Meter equipped with a charge coupled device camera. Images were then analyzed by the equipment software, and drop dimensions were used to calculate the surface tension of the solutions at the air−water interface by fitting the dimensions into the Young−Laplace equation. To validate the calibration, the surface tension of pure water (Milli-Q water with a resistivity of ∼18 MΩ·cm) was measured. A surface tension of 71.99 ± 0.04 mN/m was expected. Control groups where lipid films were hydrated in PBS were used. Surface Tension Using Rising Drop Principle. An air-tight syringe equipped with a J-shaped needle (Krüss, model: NE72, 1.001 mm diameter) was filled with octafluoropropane (C3F8) gas (Electronic Fluorocarbons, LLC, Ivyland, PA). Solutions were placed in a custom-made clear cuvette. A bubble of the C3F8 gas was formed at the tip of the J-shaped needle inside the lipid−surfactant solution containing cuvette, forming a gas bubble surrounded by the lipid and Pluronic solution. Images of the drops were acquired and analyzed as described with the previous technique. Control groups consisted of C3F8 gas bubble formed inside a solution containing the lipids mixture without Pluronic L10. Nanobubble Formulation. Lipid films were hydrated in 1 mL of L10 solution in PBS, at varying L10:lipids molar ratio, in the presence of 50 μL of glycerol (Acros Organics, NJ) at 75 °C for 30 min. The air inside of bubble vials was replaced with C3F8 gas, and vials were agitated using a Vialmix shaker (Bristol-Myers Squibb Medical Imaging, North Billerica, MA) for 45 s. To isolate the nanobubble population, the bubbles were centrifuged at 50 rcf for 5 min41 and nanobubbles were withdrawn from the bottom portion of the vial. An illustration demonstrating this fabrication process is shown in Figure 1. Particle distributions were measured by dynamic light scattering (DLS) using a Litesizer 500 (Anton Paar, Austria). Samples for DLS were measured by diluting samples 1:1000 with PBS at pH 7.4, respectively (n = 3). Nanobubble Stability Setup and Analysis. Nanobubble stability studies were performed using a clinical ultrasound (Toshiba Aplio XG, SSA-790A, Toshiba Medical Imaging Systems, Otawara-Shi, Japan) equipped with a PLT-1204BT linear transducer. Isolated nanobubbles were diluted 1:200 in PBS and were injected in a custommade agarose mold (Figure 5A). This phantom was placed directly over the transducer, and the slit was aligned with the transducer allowing the bubbles to be continuously impacted by the ultrasound field. Nanobubbles were imaged for 15 min under constant ultrasound using the following parameters: 12 MHz frequency, mechanical index of 0.1, 0.2 frame rate, 1.5 in focus depth, dynamic range of 65 dB, and

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RESULTS AND DISCUSSION A significant body of work has investigated the contributions of the microbubble shell on the stability and acoustic properties of these structures. The shell components of a static bubble must be compressible and insoluble to reduce the surface tension to near 0 to prevent bubble dissolution.45,46 In addition to contributing to bubble stability, the lipid shell properties, such as viscosity and friction damping, are critical to determining the acoustic response of a bubble.29,31,47,48 The purpose of this study was to expand on this previous work to investigate the effect of incorporating the polymer co-surfactant, Pluronic L10, to the lipid monolayer of a nanobubble on membrane cohesiveness and surface tension. Pendant Drop. To understand the effect of the incorporation of Pluronic L10 on monolayer surface tension and thus its possible effect on the stability of nanobubbles, pendant drop tensiometry was used. The surface tension at the air−water interface was measured for solutions containing L10:lipids molar ratios ranging from 0 to 0.6. After the drops of different solutions were formed, the images were acquired and the dimensions were fitted in the Young−Laplace equation. Representative pictures of the drops are presented in Figure 2A. As observed in the images, the drop dimensions are affected by the incorporation of Pluronic L10 at varying molar ratios. Specifically, the drop shape appears to elongate with an increase in Pluronic L10 molar ratio when compared to that of control solutions. The surface tension measurements of the different solutions, calculated on the basis of the pictures, are presented in Figure 2B. Even with the incorporation of Pluronic at the lowest added concentration, the surface tension decreased significantly (p < 0.005) from 67.7 ± 4.7 to 57.2 ± 0.9 mN/m, compared to that of the control group (pure lipid solution in PBS). Upon a further increase of the polymer, the surface tension continued to decrease. The highest reduction in surface tension, compared to the control group, was achieved at a L10:lipids molar ratio of 0.2, which resulted in a surface tension 9951


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considerably affected by the different surfactant concentrations. Compared to the control groups, as the amount of Pluronic L10 concentration was increased, an elongation of the drop was observed. Surface tension values were determined and are shown in Figure 3B. As with the previous methodology, the surface tension values were significantly lower than the control group and continued to decrease as the molar ratio of Pluronic to lipids increased. A minimum surface tension value of 39.2 ± 2.3 mN/m was obtained at a L10:lipids molar ratio of 0.2, representing a reduction of 35% in surface tension compared to that of the control group. After further addition of the surfactant, the measured difference in surface tension was not significant. Comparing both methodologies, we observe a significant decrease in the surface tension of the solutions with the gas interphase being C3F8 compared to air (Figure 4). This Figure 2. Surface tension measurements using the pendant drop method. (A) Drop pictures of the solutions with varying molar ratios of L10:lipids. (B) Surface tension measurements of solutions containing lipids and Pluronic L10 at varying concentrations. * Significant difference (p < 0.05). n = 6.

of 49.1 ± 0.9 mN/m, a reduction of 27.5% compared to that of the control group. The measured differences in surface tension for solutions above this concentration were not significant. This plateau could be due to the critical micellar concentration of the surfactant, which has not been reported in the literature. Pendant Bubble Rising Drop. To measure the surface tension at the perfluorocarbon gas−liquid interface, a more representative model of the nanobubble composition, C3F8 gas bubbles were formed inside a solution of varying Pluronic to lipids molar ratios. As with the pendant drop principle, images of the gas bubbles were acquired and their dimensions were fitted into the Young−Laplace equation to determine their surface tension. Representative images of the gas bubbles in the different solutions are presented in Figure 3A. Similar to the images in the previous section, gas bubble dimensions were

Figure 4. Comparison of the surface tension values obtained using both pendant drop (air−water interphase) and rising drop (C3F8− water interphase) modalities.

reduction in surface tension has been previously reported49,50 and has been assigned to the accelerated adsorption of surfaceactive excipients to the air−water interface. Results from the rising pendant bubble and the pendant drop modalities were however very comparable, having a similar trend in the reduction of the surface tension with the addition of Pluronic at different molar ratios. In both modalities, a minimum in surface tension was obtained at a L10:lipids molar ratio of 0.2. A plateau was reached upon further incorporation of the surfactant to the solutions specifically after a molar ratio of 0.4. In addition to the effects of Pluronic L10 in the decrease of the surface tension previously described, the low values can also be attributed to the primary lipid in the bubble’s outer shell, DPPC. Saturated diacyl chains, because of its low solubility and high packing order, allow the decrease of the surface tension at high compressions.32 DPPC is known to act as a surfactant, especially in the lungs where it can lower the surface tension to near 0 when combined to other phospholipids and proteins that make up the pulmonary surfactants.51,52 The mechanism by which this lipid acts as a surfactant is still debatable. One theory states the capacity of the lipid to fully pack upon quasiequilibrium compression with a Langmuir balance and “squeeze out” other components in the monolayer.51,53,54 Studies have also shown that poly(ethylene glycol) (PEG) can act as a substitute for the proteins found in pulmonary surfactants, thus helping in the reduction of surface tension.55 Nanobubble Stability Studies. To evaluate the effect of Pluronic L10 on bubble stability and correlate these results with the effect of surface tension, nanobubbles with different molar ratios were formulated. Bubbles were imaged under ultrasound in an agarose phantom for 15 min (Figure 5A). CHI

Figure 3. Surface tension measurements using the rising drop method. (A) Drop pictures of the solutions with varying molar ratios. (B) Surface tension measurements of C3F8 drops in solutions containing lipids and Pluronic L10 at varying concentrations. * Significant difference (p < 0.05). n = 6. 9952


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Figure 5. Nanobubble stability studies. (A) Schematic of custom-made agarose mold containing the nanobubbles and its alignment with the ultrasound transducer and an example of US images at 0 and after 5 min of image acquisition. (B) Representative changes in US signal intensity over time for nanobubbles with different formulations. (C) Changes in nanobubble half-life of different nanobubble formulations. (D) Dependence of nanobubble size on shell composition.

quantification data were analyzed, and the signal intensity decay over time for each formulation was plotted (Figure 5B). Nanobubbles with a L10/lipid molar ratio of 0.2 resulted in the most stable bubbles, exhibiting the lowest signal decay over time, compared to that of all other formulations. To further obtain a quantification of the nanobubble stability at the different formulations, the bubbles’ half-life was calculated (Figure 5C). An increase in the stability was observed with the increase in Pluronic L10 concentration from a molar ratio of 0.02 up to 0.2. Nanobubbles with a L10:lipids molar ratio of 0.2 had a significantly higher half-life (p < 0.05, 4.8 ± 0.2 min) than the control group (3.4 ± 0.3 min), a 29% increase. Nanobubble size was relatively unaffected by the concentration of Pluronic added (Figure 5D). No bubble signal was present for bubbles formulated with Pluronic only (without lipids, data not shown). We hypothesize that a Pluronic-only membrane may not have lateral cohesive forces necessary to stabilize the gas core or resonate at clinical ultrasound frequencies. According to the Laplace equation, it would be anticipated that a significant decrease in monolayer surface tension would result in a measurable increase in bubble stability under ultrasound. However, this trend was not completely observed. As mentioned earlier, results determined using the rising drop and pendant drop method indicated that an increase in the L10:lipids molar ratio from 0 to 0.02 significantly decreased the surface tension. However, there was no significant difference in bubble stability between these two groups. Likewise, although there was no measurable difference in surface tension when increasing the L10:lipids molar ratio from 0.4 to 0.6, this increase in L10 significantly decreased bubble stability. This inconsistency can be in part explained by the fact that both pendant drop and rising drop measure the equilibrium or static values of surface tension. However, when a bubble is stimulated by an ultrasound wave, its compressible gas expands and contracts with the applied pressure rarefaction and compression, respectively (Figure 6A).56 This expansion under ultrasound has been visualized for microbubbles with high-

Figure 6. Bubble membrane behavior under ultrasound. (A) Change in bubble diameter in response to ultrasound pressure. (B) Schematic of lipid membrane structure undergoing compression and expansion. (C) Schematic of theoretical Π−A isotherms and corresponding structure of a lipid monolayer.

speed microscopy where they were found to expand 2−4 times in radius, a 4- to 16-fold increase in surface area, depending on the applied ultrasound frequency and acoustic pressure.57 Assuming a constant monolayer lipid concentration, these rapid bubble expansion and compression result in a kinetic oscillation in the packing of surface-active agents in the monolayer, which can be thought as cycling the monolayer packing between a 9953


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surface pressure reflect a crystalline-like packing of monolayer components. Monolayers containing a L10:lipid ratio higher than 0.2 (indicated with a black arrow) were more aligned with the L10 only monolayer and demonstrated lower surface pressures at this compression (Figure 7B). As mentioned earlier, nanobubbles undergo a cycle of contraction and expansion while under ultrasound field, which causes its membrane to shift between contraction and expansion. Although it is currently technically challenging to instantaneously measure the surface tension of the membrane as it contracts and expands, the Langmuir−Blodgett data can provide some insight as to how the surface tension of the membrane changes as you compress it from liquid-crystalline (analogous to the expanded nanobubble membrane) to crystalline phases (analogous to contracted nanobubble membrane). At very low compressions, increasing L10:lipids molar ratio was shown to increase the surface pressure. However, at high compressions, increasing the L10:lipids molar ratio was shown to decrease surface pressure. Because surface pressure is inversely proportional to surface tension, these results indicate that incorporating more L10 into the nanobubble monolayer has a stabilizing effect when the membrane is expanded and a destabilizing effect when the membrane is contracted. This effect is due to the concentration-dependent influence of both the lipids and the L10 on the surface pressure. This means that microstructure of the domains and morphologies achieved by mixing these two components play a role in the viscoelastic properties of the shell layer in actual dynamic experiments. Langmuir film monolayer studies based on Brewster angle microscopy60 and surface rheology61 experiments can shed light on some of these composition−monolayer properties correlation and is related to their behavior in dynamic bubble models. Still, these results indicate a potential for optimization wherein surface pressure can be maximized for both contraction and expansion phases at an L10:lipids ratio of 0.2.

crystalline-like state and a liquid-crystalline-like state (Figure 6B,C). Understanding the change in surface tension of the membrane as it undergoes this oscillating transition can provide us a better idea of how individual components contribute to the stability of nanobubbles under ultrasound field. This kinetic monolayer behavior was thus studied using a Langmuir− Blodgett film. Π−A Isotherms. To better understand the role that Pluronic L10 may have in a mixed polymer/lipid monolayer, the behavior of L10 alone was first studied. To measure the surface activity of L10 at the bare air−water interface, a very low subphase concentration (0.34 ppb) was added to the trough. The Π−A isotherm of L10 only (Figure 7A)

Figure 7. Surface activity of Pluronic L10. (A) Π−A isotherms of Pluronic L10 and phospholipids at varying molar ratios at the air−water interface. (B) Average surface pressure at trough areas of 100 and 400 cm2.

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CONCLUSIONS In this study, the interfacial properties of nanobubble monolayers were studied to specifically evaluate the surface tension effect of the incorporation of Pluronic L10 in the monolayer through Langmuir−Blodgett isotherms and optical tensiometry. Both techniques demonstrated a decrease in surface tension with increasing concentrations of the surfactant until a maximum molar ratio. After this point, a plateau was reached with further incorporation of the surfactant suggesting that after this concentration the surfactant’s critical micelle concentration value was exceeded. We hypothesized that the reduction of surface tension achieved by the incorporation of Pluronic L10 into the nanobubble monolayer had a stabilizing effect in the bubbles through the Young−Laplace equation. To test this, nanobubbles with the varying Pluronic concentrations were developed and their stability under a clinical ultrasound was studied. Results correlated with our hypothesis, proving that the incorporation of the surfactant aids in stabilizing the bubbles through surface tension reduction and thus a reduction in the Laplace pressure. However, this effect was seen only for certain compositions and varied with monolayer structure (crystalline phase or liquid-crystalline packing). Contracted and expanded phases of the Langmuir−Blodgett analysis can be correlated with bubble oscillations within the ultrasound field. Thus, an optimal, synergistic shell composition, which is robust

demonstrated a small surface pressure transition suggesting that it stays in the expanded phase throughout compression. The high surface pressure for all trough areas is likely due to large polymer molecular weight and repulsive interactions of the PEO sections of L10.58,59 A collapse pressure of 27 mN/m at a trough area of 72 cm2 was determined for L10 polymer only. Π−A isotherms of various ratios of mixed polymer/lipid monolayers (Figure 7A) demonstrated a gradual shape transition from pure lipids to L10 only. An increase in the L10:lipids ratio corresponded to an increase in surface pressure at high trough areas (>400 cm2), indicating a decrease in surface tension with the incorporation of L10. As shown in Figure 7B, a statistically significant increase (p < 0.05) in the average surface pressure (at 400 cm2 for comparison) was measured when the L10:lipids molar ratio was increased from 0.02 to 0.2, indicating a shift from the lipid-dominated interface to an L10-dominated interface. This transition is also evident once the monolayer is compressed further. At a trough area of approximately 100 cm2, all monolayers with a L10:lipids molar ratio less than 0.2 exhibited large increases in surface pressure with small decreases in trough area, following a similar trend to that of the pure lipid monolayer. These sharp increases in 9954


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under both of these conditions, is likely to yield the most robust bubbles for in vivo applications.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Christopher Hernandez: 0000-0003-0380-7136 Rigoberto Advincula: 0000-0002-2899-4778 Author Contributions ∥

C.H. and L.N. contributed equally to this study.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Institutes of Health (F31-CA200373 and T32-EB007509 to C.H.) and the Office of the Assistant Secretary of Defense for Health Affairs, through the Prostate Cancer Research Program under Award No. W81XWH-16-1-037I (to A.A.E.) in addition to the PostBaccalaureate Research Education Program grant (R25GM075207 to L.N.). We also acknowledge additional support from the Case Comprehensive Cancer Center P30CA043703 in the form of a pilot grant. Views and opinions of, and endorsements by the author(s) do not reflect those of the National Institutes of Health or of the Department of Defense.

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