Role of Surface Tension in Gas Nanobubble Stability Under Ultrasound

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The Role of Surface Tension in Gas Nanobubble Stability Under Ultrasound Christopher Hernandez, Lenitza Nieves, Al C. de Leon, Rigoberto Advincula, and Agata A. Exner ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19755 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 3, 2018

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ACS Applied Materials & Interfaces

The Role of Surface Tension in Gas Nanobubble Stability Under Ultrasound

Christopher Hernandez1*, Lenitza Nieves2*, Al C. de Leon2, Rigoberto Advincula3, and Agata A. Exner2,1.

Departments of Biomedical Engineering1, Radiology2, and Macromolecular Science and Engineering3, Case Western Reserve University, Cleveland, OH, USA *These authors contributed equally to this study.

Christopher Hernandez

[email protected]

Lenitza Nieves

[email protected]

Al C. de Leon

[email protected]

Rigoberto Advincula

[email protected]

To Whom Correspondence Should Be Addressed: Dr. Agata A. Exner Professor of Radiology and Biomedical Engineering Case Western Reserve University 10900 Euclid Ave. Cleveland, OH 44106 USA [email protected] Key words: Ultrasound contrast agents, Microbubbles, Nanobubbles, Surface tension, Langmuir Blodgett, Pendant drop

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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 sub-micron 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 interaction with nanoparticles should not be readily detectable at clinicallyrelevant 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 bubble-stabilizing 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.


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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. While 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 lesions1. 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. While 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 a sub-micron 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 (EPR) effect 5-6. Maximal accumulation is typically seen for particles less than 200 nm7. However, this effect is highly heterogeneous and has been found to depend on many factors including tumor type, nanoparticle shape8 and particle mechanical properties9 studies have shown that bacteria as large as 1000 nm10 to accumulate in tumors. While sub-micron 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 controversy11. This is in part due to the size-dependent stability of an uncoated nanobubble that is predicted by the Laplace pressure. As shown in equation (1) this pressure (P) is dependent on the bubble radius (R) and the surface tension (σ). ∆ܲ = ܲ௜௡௦௜ௗ௘ − ܲ௢௨௧௦௜ௗ௘ =

ଶఙ ோ

(1)

As the radius decreases to the nanometer range, this pressure difference increases to several atmospheres, making the nanobubbles thermodynamically unstable. Additionally, bubble stability is related to the counteractive forces of the partial pressures of dissolved gasses in the surrounding fluid. A more comprehensive predictive model for bubble stability, proposed by Epstein and Plesset in 1950, predicts that an uncoated sub-micron bubble, in a gas saturated medium would have a lifetime of less than one second12. Contrary to what these two equations predict, naturally occurring oceanic nanobubbles have been observed to be stable for over 20 hours13. These bubbles were proven to be



coated with naturally occurring surfactants that reduced their surface tension.

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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 size13. In order 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 gas filled pockets on a surface with a spherical cap (for recent reviews see 14-16). Surface nanobubbles were initially discovered and imaged using atomic force microscopy (AFM), and their existence was later confirmed with other techniques15. Various theories including, contamination of their surface17, the dynamic equilibrium theory18, and pinning19 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 sub-micron contrast agents for their use in ultrasound-guided cancer detection and theranostics20-21. These formulations have ranged from echogenic liposomes (ELIPs)22-23 and phase-shifting nanoemulsions24 to polymeric or lipid-coated nanobubbles25-28. These particles act as echo-enhancers due to high acoustic impedance difference between the gas and blood. While they have shown promise in pre-clinical 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 frequencies29, which ultimately harms 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 processes30-33}. 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 Pluronic34-37. Pluronic is a non-ionic triblock copolymer composed of repeating subunits of polyethylene oxide (PEO) and polypropylene 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 airwater interface by folding into a V-shape, with the center PPO chains interacting with the hydrophobic lipid acyl chains and the outer PPO segments interacting with the hydrophilic lipid polar head groups38-40. Evidence of a gas core in these constructs has been demonstrated with cryo-EM41 and using the Resonant Mass Measurement technique42. Additionally, these bubbles have strong ultrasound contrast enhancement which is consistent with a compressible gas core as opposed to oil droplets42-43.


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While 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 agent. We hypothesize that the incorporation of Pluronic into the formulation stabilizes nanobubbles by imparting additional barrier robustness to counteract applied pressure as consequence of the reduction in its membrane’s surface tension. Numerical simulations of nanobubble non-linear 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 frequencies44. In the current report, we investigated the effects of varying Pluronic-lipid ratios in the bubble shell on nanobubble behavior in vitro. Specifically, the impact of shell composition on membrane equilibrium surface tension was investigated using optical tensiometry using both pendant drop and rising drop principles. Kinetic or quasi-equilibrium surface tension, which better describes bubble stability under an ultrasound field was investigated via Langmuir Blodgett analysis. Experimental Materials The lipids DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DPPA (1,2 Dipalmitoyl-snGlycero-3-Phosphate), and DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) were obtained from Corden Pharma (Switzerland), and mPEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)-2000] (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 1mL of a L10 solution in 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 minutes. 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 the drops were acquired, in triplicate, using a KSV CAM 200 Optical Contact Angle Meter equipped with a CCD 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 ~18MΩ·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, USA, model: NE72, 1.001 mm diameter) was filled with octaflouropropane (C3F8) gas (Electronic Fluorocarbons, LLC., Ivyland, PA, USA). Solutions were placed in a custom-made clear cuvette. A bubble of the C3F8 gas was formed in the tip of the J-shaped needle inside of the lipids-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 (New Jersey, USA) at 75°C for 30 minutes. 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, USA) for 45 seconds. To isolate the nanobubble population, the bubbles were centrifuged at 50 rcf for 5 minutes41, 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).


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Figure 1. Illustration demonstrating the fabrication process for Pluronic nanobubbles

Nanobubble Stability Setup and Analysis Nanobubble stability studies were performed using a clinical ultrasound (Toshiba Aplio XG, SSA790A, 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 custom-made 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 minutes under constant ultrasound using the following parameters: 12 MHz Frequency, mechanical index (MI) of 0.1, 0.2 frame rate, 1.5 in focus depth, dynamic range of 65 dB and 2D gain of 70 dB, 15 minutes. 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). Ultra-Pure water (resistivity ~ 18MΩ·cm) was used as the subphase. Lipid-surfactant solutions were made by dissolving the DPPC, DPPA, DPPE 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 lipidsurfactant solution was spread throughout the trough. The trough equilibrated for 10 minutes 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 are 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. 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 in order to reduce the surface tension to near zero in order to prevent bubble dissolution45-46. 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 bubble29, 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 properties 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


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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 control solutions. The surface tension measurements of the different solutions, calculated based on the pictures, are presented in Figure 2B. Even with the incorporation of Pluronic at the lowest added concentration, the surface tension decreased significantly (p

Role of Surface Tension in Gas Nanobubble Stability Under Ultrasound

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