Brandon Helfield

In situ conversion of porphyrin microbubbles to nanoparticles for multimodality imaging

Converting nanoparticles or monomeric compounds into larger supramolecular structures by endogenous or external stimuli is increasingly popular because these materials are useful for imaging and treating diseases. However, conversion of microstructures to nanostructures is less common. Here, we show the conversion of microbubbles to nanoparticles using low-frequency ultrasound. The microbubble consists of a bacteriochlorophyll-lipid shell around a perfluoropropane gas. The encapsulated gas provides ultrasound imaging contrast and the porphyrins in the shell confer photoacoustic and fluorescent properties. On exposure to ultrasound, the microbubbles burst and form smaller nanoparticles that possess the same optical properties as the original microbubble. We show that this conversion is possible in tumour-bearing mice and could be validated using photoacoustic imaging. With this conversion, our microbubble can potentially be used to bypass the enhanced permeability and retention effect when delivering drugs to tumours.

Methylene blue microbubbles as a model dual-modality contrast agent for ultrasound and activatable photoacoustic imaging

Abstract. Ultrasound and photoacoustic imaging are highly complementary modalities since both use ultrasonic detection for operation. Increasingly, photoacoustic and ultrasound have been integrated in terms of hardware instrumentation. To generate a broadly accessible dual-modality contrast agent, we generated microbubbles (a standard ultrasound contrast agent) in a solution of methylene blue (a standard photoacoustic dye). This MB2 solution was formed effectively and was optimized as a dual-modality contrast solution. As microbubble concentration increased (with methylene blue concentration constant), photoacoustic signal was attenuated in the MB2 solution. When methylene blue concentration increased (with microbubble concentration held constant), no ultrasonic interference was observed. Using an MB2 solution that strongly attenuated all photoacoustic signal, high powered ultrasound could be used to burst the microbubbles and dramatically enhance photoacoustic contrast (>800-fold increase), providing a new method for spatiotemporal control of photoacoustic signal generation.

Biophysical insight into mechanisms of sonoporation

Significance Gas-filled microbubbles physically oscillate in an ultrasound field and have been shown to potentiate the delivery of therapeutic payloads. The biophysical mechanisms by which vibrating microbubbles stimulate macromolecule uptake across the cell membrane, however, remain unknown. With a coupled microscopy system capable of bright-field imaging up to 25 million frames per second, we show correlations between microsecond-scale bubble oscillations and second-to-minute–scale macromolecule diffusion, uniquely highlighting that microbubble-induced shear stress is a threshold indicator for membrane pore generation. Further insight into membrane reorganization using real-time confocal microscopy demonstrates that ultrasound-triggered microbubbles create resealing pores through both layers of cellular membrane, and gaps between confluent cells. This work presents mechanistic, biophysical insight into sonoporation as a tool for local delivery of therapeutic macromolecules. This study presents a unique approach to understanding the biophysical mechanisms of ultrasound-triggered cell membrane disruption (i.e., sonoporation). We report direct correlations between ultrasound-stimulated encapsulated microbubble oscillation physics and the resulting cellular membrane permeability by simultaneous microscopy of these two processes over their intrinsic physical timescales (microseconds for microbubble dynamics and seconds to minutes for local macromolecule uptake and cell membrane reorganization). We show that there exists a microbubble oscillation-induced shear-stress threshold, on the order of kilopascals, beyond which endothelial cellular membrane permeability increases. The shear-stress threshold exhibits an inverse square-root relation to the number of oscillation cycles and an approximately linear dependence on ultrasound frequency from 0.5 to 2 MHz. Further, via real-time 3D confocal microscopy measurements, our data provide evidence that a sonoporation event directly results in the immediate generation of membrane pores through both apical and basal cell membrane layers that reseal along their lateral area (resealing time of ∼<2 min). Finally, we demonstrate the potential for sonoporation to indirectly initiate prolonged, intercellular gaps between adjacent, confluent cells (∼>30–60 min). This real-time microscopic approach has provided insight into both the physical, cavitation-based mechanisms of sonoporation and the biophysical, cell-membrane–based mechanisms by which microbubble acoustic behaviors cause acute and sustained enhancement of cellular and vascular permeability.

Fluid Viscosity Affects the Fragmentation and Inertial Cavitation Threshold of Lipid-Encapsulated Microbubbles

Ultrasound and microbubble optimization studies for therapeutic applications are often conducted in water/saline, with a fluid viscosity of 1 cP. In an in vivo context, microbubbles are situated in blood, a more viscous fluid (∼4 cP). In this study, ultrahigh-speed microscopy and passive cavitation approaches were employed to investigate the effect of fluid viscosity on microbubble behavior at 1 MHz subject to high pressures (0.25-2 MPa). The propensity for individual microbubble (n = 220) fragmentation was found to significantly decrease in 4-cP fluid compared with 1-cP fluid, despite achieving similar maximum radial excursions. Microbubble populations diluted in 4-cP fluid exhibited decreased wideband emissions (up to 10.2 times), and increasingly distinct harmonic emission peaks (e.g., ultraharmonic) with increasing pressure, compared with those in 1-cP fluid. These results suggest that in vitro studies should consider an evaluation using physiologic viscosity perfusate before transitioning to in vivo evaluations.

The influence of compliant boundary proximity on the fundamental and subharmonic emissions from individual microbubbles

The proximity of a solid-liquid boundary has been theoretically predicted to affect nonlinear microbubble emissions, but to date there has been no experimental validation of this effect. In this study, individual microbubbles (n = 15) were insonicated at f = 11 MHz as a function of offset distance from a compliant (agarose) planar boundary by employing an optical trapping apparatus. It was found that fundamental scattering increases while subharmonic scattering decreases as the microbubble approaches the boundary. Although a microbubble-boundary model can predict the qualitative trends observed for a subset of encapsulation properties, further modeling efforts are required to completely model compliant boundary-microbubble interactions.

Individual lipid encapsulated microbubble radial oscillations: Effects of fluid viscosity

Ultrasound-stimulated microbubble dynamics have been shown to be dependent on intrinsic bubble properties, including size and shell characteristics. The effect of the surrounding environment on microbubble response, however, has been less investigated. In particular, microbubble optimization studies are generally conducted in water/saline, characterized by a 1 cP viscosity, for application in the vasculature (i.e., 4 cP). In this study, ultra-high speed microscopy was employed to investigate fluid viscosity effects on phospholipid encapsulated microbubble oscillations at 1 MHz, using a single, eight-cycle pulse at peak negative pressures of 100 and 250 kPa. Microbubble oscillations were shown to be affected by fluid viscosity in a size- and pressure-dependent manner. In general, the oscillation amplitudes exhibited by microbubbles between 3 and 6 μm in 1 cP fluid were larger than in 4 cP fluid, reaching a maximum of 1.7-fold at 100 kPa for microbubbles 3.8 μm in diameter and 1.35-fold at 250 kPa for microbubbles 4.8 μm in diameter. Simulation results were in broad agreement at 250 kPa, however generally underestimated the effect of fluid viscosity at 100 kPa. This is the first experimental demonstration documenting the effects of surrounding fluid viscosity on microbubble oscillations, resulting in behavior not entirely predicted by current microbubble models.

Insight into the plasma membrane resealing and calcium signaling dynamics of sonoporation

Ultrasound (US) and microbubble (MB) treatment has been shown to be a promising approach for localized, non-viral gene delivery. The attributes of MB-target cell interactions that facilitate payload delivery across cell membranes and into extravascular cells, and hence strategies to optimize this platform, remain poorly understood. The objective of this work is to gain insight into the extent of sonoporation by assessing cell membrane dynamics and Ca2+ signaling, both known factors in cell perforation repair, and the role of gap junctional communication (GPC).

Ultrafast frame rate microscopy of microbubble oscillations: Current studies employing the UPMC-Cam

Ultrasound-stimulated microbubbles are clinically employed for diagnostic imaging and have been shown to be a feasible therapeutic strategy for localized drug and gene delivery applications. In order to investigate microbubble oscillation dynamics, the University of Pittsburgh Medical Center has developed the UPMC-Cam—an ultrafast imaging system capable of recording 128 frames at up to 25 million frames per second, one of only two in the world currently in use. Current studies include elucidating the effect of fluid viscosity on microbubble behavior. At lower pressures (0.1–0.25 MPa), an increase in fluid viscosity from 1 to 4 cP alters the fundamental and second-harmonic oscillation amplitudes in a bubble size-dependent manner, related to resonance. At higher pressures (0.5–1.5 MPa), a significant decrease in the propensity for microbubble fragmentation was observed in the more viscid fluid environment. Additionally, studies aimed at gaining physical insights into sonoporation have been conducted in whic...

The effect of boundary proximity on the fundamental and subharmonic emissions from individual microbubbles at higher frequencies

It is recognized that the proximity of a boundary can influence the dynamic behavior of acoustically stimulated microbubbles. In a biomedical ultrasound context, this is relevant to molecular imaging with targeted microbubbles, and when microbubbles are near vessel walls or contained within microvessels. Theoretical models have recently been developed to examine these effects, but experimental work has been more limited and primarily focused on the assessment of resonant frequency effects rather than its impact on nonlinear behavior, which is perhaps more relevant to imaging applications. Understanding this behavior is important to improving microbubble detection and for the quantitative interpretation of contrast images. With the use of an optical trap, this study experimentally investigates the effect of boundary proximity (0 to 200 µm) and boundary stiffness (Opticell and agarose) on fundamental and subharmonic emissions from individual Definity and MicroMarker bubbles at 11 MHz. The scattered pressure...

Subharmonic behavior of targeted and untargeted lipid encapsulated microbubbles at high ultrasound frequencies.

Molecular imaging with ultrasound contrast agents (microbubbles) has recently gained interest as a feasible technique for disease‐specific imaging, with applications ranging from intravascular ultrasound to small animal imaging. The attachment of targeting ligands to the microbubble shell enables a selective accumulation of bound microbubbles around a target site. The ability, however, to differentiate between the nonlinear signal from bound microbubbles and from unbound, circulating agent still remains a challenge. This study conducts a size‐per‐size comparison of the acoustic nonlinear response of individual streptavidin‐coated MicroMarker microbubbles either bound (BMM) or adjacent (UBMM) to a compliant agarose/biotin gel surface. Bubbles were optically sized and insonified at 25 MHz over a range of pressures and pulse bandwidths. The subharmonic (nonlinear) response between unbound (n = 24) and bound (n = 29) bubbles was found to differ significantly, with UBMM bubbles having a higher propensity to in...