Hydrostatic Pressurization of Lung Surfactant Microbubbles: Observation of a Strain-Rate Dependent Elasticity

The microbubble offers a unique platform to study lung surfactant mechanics at physiologically relevant geometry and length scale. In this study, we compared the response of microbubbles (∼15 μm initial radius) coated with pure dipalmitoyl-phosphatidylcholine (DPPC) versus naturally derived lung surfactant (SURVANTA) when subjected to linearly increasing hydrostatic pressure at different rates (0.5–2.3 kPa/s) at room temperature. The microbubbles contained perfluorobutane gas and were submerged in buffered saline saturated with perfluorobutane at atmospheric pressure. Bright-field microscopy showed that DPPC microbubbles compressed spherically and smoothly, whereas SURVANTA microbubbles exhibited wrinkling and smoothing cycles associated with buckling and collapse. Seismograph analysis showed that the SURVANTA collapse amplitude was constant, but the collapse rate increased with the pressurization rate. An analysis of the pressure–volume curves indicated that the dilatational elasticity increased during compression for both shell types. The initial dilatational elasticity for SURVANTA was nearly twice that of DPPC at higher pressurization rates (>1.5 kPa/s), producing a pressure drop of up to 60 kPa across the film prior to condensation of the perfluorobutane core. The strain-rate dependent stiffening of SURVANTA shells likely arises from their composition and microstructure, which provide enhanced in-plane monolayer rigidity and lateral repulsion from surface-associated collapse structures. Overall, these results provide new insights into lung surfactant mechanics and collapse behavior during compression

Fluorocarbon Nanodrops as Acoustic Temperature Probes

This work investigated the use of superheated fluorocarbon nanodrops for ultrasound thermal imaging and the use of mixed fluorocarbons for tuning thermal and acoustic thresholds for vaporization. Droplets were fabricated by condensing phospholipid-coated microbubbles containing C₃F₈ and C₄F₁₀ mixed at various molar ratios. Vaporization temperatures first were measured in a closed system by optical transmission following either isothermal pressure release or isobaric heating. The vaporization temperature was found to depend linearly on the percentage of C₄F₁₀ in the droplet core, indicating excellent tunability under these fluorocarbon-saturated conditions. Vaporization temperatures were then measured in an open system using contrast-enhanced ultrasound imaging, where it was found that the mixed droplets behaved like pure C₄F₁₀ drops. Additionally, the critical mechanical index for vaporization was measured at the limits of therapeutic hyperthermia (37 and 60 °C), and again the mixed droplets were found to behave like pure C₄F₁₀ drops. These results suggested that C₃F₈ preferentially dissolves out of the droplet core in open systems, as shown by a simple mass transfer model of multicomponent droplet dissolution. Finally, proof-of-concept was shown that pure C₄F₁₀ nanodrops can be used as an acoustic temperature probe. Overall, these results not only demonstrate the potential of superheated fluorocarbon emulsions for sonothermetry but also point to the limits of tunability for fluorocarbon mixtures owing to preferential release of the more soluble species to the atmosphere

Thermal Activation of Superheated Lipid-Coated Perfluorocarbon Drops

This study explored the thermal conditions necessary for the vaporization of superheated perfluorocarbon nanodrops. Droplets C₃F₈ and C₄F₁₀ coated with a homologous series of saturated diacyl­phosphatidyl­cholines were formed by condensation of 4 μm diameter microbubbles. These drops were stable at room temperature and atmospheric pressure, but they vaporized back into microbubbles at higher temperatures. The vaporization transition was measured as a function of temperature by laser light extinction. We found that C₃F₈ and C₄F₁₀ drops experienced 90% vaporization at 40 and 75 °C, respectively, near the theoretical superheat limits (80–90% of the critical temperature). We therefore conclude that the metastabilty of these phase-change agents arises not from the droplet Laplace pressure altering the boiling point, as previously reported, but from the metastability of the pure superheated fluid to homogeneous nucleation. The rate of C₄F₁₀ drop vaporization was quantified at temperatures ranging from 55 to 75 °C, and an apparent activation energy barrier was calculated from an Arrhenius plot. Interestingly, the activation energy increased linearly with acyl chain length from C14 to C20, indicating that lipid interchain cohesion plays an important role in suppressing the vaporization rate. The vaporized drops (microbubbles) were found to be unstable to dissolution at high temperatures, particularly for C14 and C16. However, proper choice of the fluorocarbon and lipid species provided a nanoemulsion that could undergo at least ten reversible condensation/vaporization cycles. The vaporization properties presented in this study may facilitate the engineering of tunable phase-shift particles for diagnostic imaging, targeted drug delivery, tissue ablation, and other applications

Condensation Phase Diagrams for Lipid-Coated Perfluorobutane Microbubbles

The goal of this study was to explore the thermodynamic conditions necessary to condense aqueous suspensions of lipid-coated gas-filled microbubbles into metastable liquid-filled nanodrops as well as the physicochemical mechanisms involved with this process. Individual perfluorobutane microbubbles and their lipid shells were observed as they were pressurized at 34.5 kPa s^–1 in a microscopic viewing chamber maintained at temperatures ranging from 5 to 75 °C. The microbubbles contracted under pressure, ultimately leading to either full dissolution or microbubble-to-nanodrop condensation. Temperature–pressure phase diagrams conveying condensation and stability transitions were constructed for microbubbles coated with saturated diacylphosphatidylcholine lipids of varying acyl chain length (C16 to C24). The onset of full dissolution was shifted to higher temperatures with the use of longer acyl chain lipids or supersaturated media. Longer chain lipid shells resisted both dissolution of the gas core and mechanical compression through a pronounced wrinkle-to-fold collapse transition. Interestingly, the lipid shell also provided a mechanical resistance to condensation, shifting the vapor-to-liquid transition to higher pressures than for bulk perfluorobutane. This result indicated that the lipid shell can provide a negative apparent surface tension under compression. Overall, the results of this study will aid in the design and formulation of vaporizable fluorocarbon nanodrops for various applications, such as diagnostic ultrasound imaging, targeted drug delivery, and thermal ablation

Monodispersity Increases Adhesion Efficiency and Specificity for Ultrasound-Targeted Microbubbles

Ultrasound molecular imaging with targeted microbubbles (MBs) can be used to noninvasively diagnose, monitor, and study the progression of different endothelial-associated diseases. Acoustic radiation force (Frad) can initiate and enhance MB adhesion at the target site. The goal of this study was to elucidate the effects of various MB parameters on Frad targeting. Monodisperse or polydisperse MBs with the immune-stealth cloaked (buried)-ligand architecture were conjugated with targeting RGD or nonspecific isotype control RAD peptides and then pumped through an αvβ3 integrin-coated microvessel phantom at a wall shear stress of 3.5 dyn/cm2. Targeting was assessed by measuring MB attachment for varying Frad time and frequency, as well as MB concentration and size distribution. We first confirmed that primary Frad is necessary to target the cloaked-ligand MBs. MB targeting increased monotonically with αvβ3 integrin density and Frad time. MB attachment and, to a lesser extent specificity, also increased when driven by Frad near resonance. MB targeting increased with MB concentration, although a shift in behavior was observed with increasing MB–MB interactions and aggregations forming from secondary Frad effects as MB concentration was increased. These secondary Frad effects reduced targeting specificity. Finally, after having validated our approach by testing different parameters with the appropriate controls, we then determined the effects of monodispersity on adhesion efficiency and specific targeting. We observed that both MB targeting efficiency and specificity were greatly enhanced for monodisperse vs polydisperse MBs. Analysis of videomicroscopy images indicated that secondary Frad effects may have disproportionally inhibited targeting of polydisperse MBs. In conclusion, our in vitro results indicate that monodisperse MBs driven near resonance and at a low concentration (∼106 MB/mL) can be used to maximize the adhesion efficiency (up to 88%) and specificity of RGD–MB targeting

Single Microbubble Measurements of Lipid Monolayer Viscoelastic Properties for Small-Amplitude Oscillations

Lipid monolayer rheology plays an important role in a variety of interfacial phenomena, the physics of biological membranes, and the dynamic response of acoustic bubbles and drops. We show here measurements of lipid monolayer elasticity and viscosity for very small strains at megahertz frequency. Individual plasmonic microbubbles of 2–6 μm radius were photothermally activated with a short laser pulse, and the subsequent nanometer-scale radial oscillations during ring-down were monitored by optical scatter. This method provided average dynamic response measurements of single microbubbles. Each microbubble was modeled as an underdamped linear oscillator to determine the damping ratio and eigenfrequency, and thus the lipid monolayer viscosity and elasticity. Our nonisothermal measurement technique revealed viscoelastic trends for different lipid shell compositions. We observed a significant increase in surface elasticity with the lipid acyl chain length for 16 to 20 carbons, and this effect was explained by an intermolecular forces model that accounts for the lipid composition, packing, and hydration. The surface viscosity was found to be equivalent for these lipid shells. We also observed an anomalous decrease in elasticity and an increase in viscosity when increasing the acyl chain length from 20 to 22 carbons. These results illustrate the use of a novel nondestructive optical technique to investigate lipid monolayer rheology in new regimes of frequency and strain, possibly elucidating the phase behavior, as well as how the dynamic response of a microbubble can be tuned by the lipid intermolecular forces

Monodispersity Increases Adhesion Efficiency and Specificity for Ultrasound-Targeted Microbubbles

Ultrasound molecular imaging with targeted microbubbles (MBs) can be used to noninvasively diagnose, monitor, and study the progression of different endothelial-associated diseases. Acoustic radiation force (Frad) can initiate and enhance MB adhesion at the target site. The goal of this study was to elucidate the effects of various MB parameters on Frad targeting. Monodisperse or polydisperse MBs with the immune-stealth cloaked (buried)-ligand architecture were conjugated with targeting RGD or nonspecific isotype control RAD peptides and then pumped through an αvβ3 integrin-coated microvessel phantom at a wall shear stress of 3.5 dyn/cm2. Targeting was assessed by measuring MB attachment for varying Frad time and frequency, as well as MB concentration and size distribution. We first confirmed that primary Frad is necessary to target the cloaked-ligand MBs. MB targeting increased monotonically with αvβ3 integrin density and Frad time. MB attachment and, to a lesser extent specificity, also increased when driven by Frad near resonance. MB targeting increased with MB concentration, although a shift in behavior was observed with increasing MB–MB interactions and aggregations forming from secondary Frad effects as MB concentration was increased. These secondary Frad effects reduced targeting specificity. Finally, after having validated our approach by testing different parameters with the appropriate controls, we then determined the effects of monodispersity on adhesion efficiency and specific targeting. We observed that both MB targeting efficiency and specificity were greatly enhanced for monodisperse vs polydisperse MBs. Analysis of videomicroscopy images indicated that secondary Frad effects may have disproportionally inhibited targeting of polydisperse MBs. In conclusion, our in vitro results indicate that monodisperse MBs driven near resonance and at a low concentration (∼106 MB/mL) can be used to maximize the adhesion efficiency (up to 88%) and specificity of RGD–MB targeting

Experimental procedures.

<p>a) Schematic of the experimental set-up used for in-vivo hypoxia modulation measurements using the Zenascope system. b) Schematic of tumor volume assessment via B-mode ultrasound imaging. Two cross-sectional images were acquired and lengths a, b and c were used to calculate tumor volume. c) Radiotherapy pre and post-imaging experimental protocol.</p

A single oxygen microbubble administration alone does not influence tumor control.

<p>No significant difference was found between the no treatment and oxygen microbubble group in the absence of any radiotherapy (n = 4 per group). Box-and-whisker plots represent all data from the No treatment and OMB alone controls.</p

Individual datapoints for radiotherapy tumor control times (in days) stratified by matched initial tumor size for each treatment group, showing RT effect size depends on initial tumor volume.

<p>Individual datapoints for radiotherapy tumor control times (in days) stratified by matched initial tumor size for each treatment group, showing RT effect size depends on initial tumor volume.</p