Aaron F. H. Lum

Optical and acoustical dynamics of microbubble contrast agents inside neutrophils.

Acoustically active microbubbles are used for contrast-enhanced ultrasound assessment of organ perfusion. In regions of inflammation, contrast agents are captured and phagocytosed by activated neutrophils adherent to the venular wall. Using direct optical observation with a high-speed camera and acoustical interrogation of individual bubbles and cells, we assessed the physical and acoustical responses of both phagocytosed and free microbubbles. Optical analysis of bubble radial oscillations during insonation demonstrated that phagocytosed microbubbles experience viscous damping within the cytoplasm and yet remain acoustically active and capable of large volumetric oscillations during an acoustic pulse. Fitting a modified version of the Rayleigh-Plesset equation that describes mechanical properties of thin shells to optical radius-time data of oscillating bubbles provided estimates of the apparent viscosity of the intracellular medium. Phagocytosed microbubbles experienced a viscous damping approximately sevenfold greater than free microbubbles. Acoustical comparison between free and phagocytosed microbubbles indicated that phagocytosed microbubbles produce an echo with a higher mean frequency than free microbubbles in response to a rarefaction-first single-cycle pulse. Moreover, this frequency increase is predicted using the modified Rayleigh-Plesset equation. We conclude that contrast-enhanced ultrasound can detect distinct acoustic signals from microbubbles inside of neutrophils and may provide a unique tool to identify activated neutrophils at sites of inflammation.

Dynamic regulation of LFA-1 activation and neutrophil arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow

Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate β2 integrin (CD18), deemed a critical step in facilitating cell capture and arrest under the shear force of blood flow. A conformational switch in the I domain allosteric site (IDAS) and in CD18 regulates LFA-1 affinity for endothelial ligands including intercellular adhesion molecule 1 (ICAM-1). We examined the dynamics of CD18 activation in terms of the efficiency of neutrophil capture of ICAM-1, and we correlated this with the membrane topography of 327C, an antibody that recognizes the active conformation of CD18 I-like domain. Adhesion increased in direct proportion to chemotactic stimulus rising 7-fold over a log range of interleukin-8 (IL-8). A threshold dose of ∼75 pm IL-8, corresponding to ligation of only ∼10–100 receptors, was sufficient to activate ∼20,000 CD18 and a rapid boost in the capture efficiency on ICAM-1. This was accompanied by a rapid redistribution of active LFA-1, but not Mac-1, into membrane patches, a necessary component for optimum adhesion efficiency. Shear-resistant arrest on a monolayer of ICAM-1 was reversed within minutes of chemotactic stimulation correlating with a shift from high to low affinity CD18 and dispersal of patches of active CD18. Mobility of active CD18 into high avidity patches was dependent on phosphatidylinositol 3-kinase activity and not F-actin polymerization. The data reveal that the number of chemotactic receptors bound and the topography and lifetime of high affinity LFA-1 tightly regulate the efficiency of neutrophil capture on ICAM-1.

Ultrasound contrast agents phagocytosed by neutrophils demonstrate acoustic activity

Ultrasound contrast agents are microbubbles composed of a thin lipid or albumin shell filled with air or a high molecular weight gas. These microbubbles are used for contrast-enhanced ultrasound (CEU) assessment of organ perfusion. In regions of inflammation, microbubbles are phagocytosed intact by activated neutrophils adherent to the venular wall. The authors hypothesized that microbubbles remain acoustically active following phagocytosis. Accordingly, they assessed the physical responses of both phagocytosed and free microbubbles by direct microscopic observation during delivery of repetitive single pulses of ultrasound at various acoustic pressures. Insonation results in oscillation in the bubbles volume. Microbubbles were optically recorded during insonation with a high-speed imaging system and diameter-time curves were analyzed to determine the effect of phagocytosis. Phagocytosed microbubbles retained their acoustic activity, although the intracellular environment increased viscoelastic damping experienced by microbubbles. With a pulse of high acoustic intensity (>1 MPa), phagocytosed microbubbles expanded up to 500% of their initial radii, which occasionally resulted in neutrophil rupture. Primary radiation force displaced phagocytosed microbubbles a distance of 100 microns with an acoustic pressure of -240 kPa and a pulse repetition frequency of 10 kHz, thus providing further evidence of acoustic activity. The authors conclude that phagocytosed microbubbles exhibit viscoelastic damping and yet are susceptible to acoustic destruction. They can generate non-linear echoes on the same order of magnitude as free microbubbles. These results indicate that CEU may be used to identify and assess regions of inflammation by detecting acoustic signals from microbubbles that are phagocytosed by activated neutrophils. In addition, the rapid expansion of a microbubble at high acoustic pressure may present a means to rupture a neutrophil or drug capsule at a specific site, resulting in delivery of a drug.

1B-3 Ultrasound Radiation Force Enables Targeted Deposition of Molecularly Targeted Nanoparticles Loaded on Microbubbles Under Flow Conditions

Novel drug delivery vehicles that specifically target using ultrasound radiation force (USRF) and molecular interactions are presented. The first model vehicle consists of commercially available Neutravidin fluorescent nanobeads bound directly to the biotinylated lipid shells of preformed microbubbles. USRF was used to deflect the vehicle from the center of flow to a tube surface in order to facilitate molecular interactions and induce adhesion. In order to reduce acoustic reflections, a cellulose tube was used as the substrate. At wall shear stress levels commensurate with venous and arterial flow, USRF (1.3 sec pulse at 3 MHz and 150 kPa peak negative pressure (PNP)) was used to direct these model vehicles to the biotinylated cellulose tube surface. Subsequent high-pressure pulses (three 5-cycle pulses at 1.5 MHz and 1.1 MPa PNP) fragmented the carrier, and biotin-Neutravidin interactions induced deposition of the nanobeads on the wall. Targeting of nanobeads to the cellulose tube was molecularly specific and dependent on, in order of importance, vehicle concentration, wall shear stress, nanobead size, and insonation time. The second vehicle takes the next steps to molecularly target the nanoparticles to biologically-relevant receptors/ligands and change the fluorescence emission to the near infrared to allow for in vivo imaging. This is accomplished by attaching Peptide LLP2A, which specifically targets integrin alpha4beta1 expressed on angiogenic vessels, to commercially available quantum dots which emit at 800 nm. These new vehicles specifically adhere to the MOLT-4 cell line which highly expresses integrin alpha4beta1, while vehicles without the specific peptide had minimal binding. This versatile method of delivery is shown to enable targeted deposition of nanoparticles in shear flow and could be used as a diagnostic tool to optically observe diseased areas as well as modified to carry therapeutic agents for controlled release in targeted delivery applications

Kinetics of LFA-1 binding to ICAM-1 studied in a cell-free system

Neutrophil capture on inflamed endothelium is controlled by dynamic regulation of the integrin CD11a/CD18 (LFA-1). Small molecules, antibodies, and certain divalent cations binding to specific epitopes on the integrin are able to stabilize either a closed (low affinity) or open (high affinity) state. To determine the relationship between LFA-1 conformation and affinity for ICAM-1 we assembled a cell-free system consisting of CD11a/CD18 heterodimer adhered to latex microspheres. The kinetics of dimeric ICAM-1 binding to the LFA-1 on the microspheres was measured via flow cytometry and a real time conformational shift into a lower affinity state was observed by addition of a small molecule inhibitor.

Enhanced drug delivery with ultrasound and engineered delivery vehicles

We demonstrate that local drug delivery can be achieved by ultrasound, combined with engineered delivery vehicles, where the vehicles have a diameter on the order of nanometers to microns. Delivery vehicles can be created from microbubbles with a thickened shell or a lipid shell decorated with drugs, genes, or nanoparticles. Alternatively, liquid‐filled nanoparticles can be employed to carry the desired compound. Ultrasound can deflect these vehicles from the center of the flowstream, can fragment the vehicle releasing its contents, and may enhance the uptake of the particle or its contents by cells in the desired region. The ultrasonic mechanisms behind these changes are summarized. The addition of targeting ligands to the shell to improve target specificity is also explored. Methods to measure the effectiveness of local drug delivery, including correlative imaging modalities, binding assays, and cytotoxicity assays, will be described. [The support of NIH CA 103828 is gratefully acknowledged.]