A novel drug delivery vehicle that specifically targets using ultrasound radiation force (USRF) and biotin-avidin interactions is presented. Model vehicles consist of avidinated 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 receptor-ligand mediated adhesion. At wall shear stress levels commensurate with venous and arterial flow, USRF was used to direct the vehicles to a biotinylated tube surface. Subsequent high-pressure pulses fragmented the carrier, and molecular interactions induced deposition of the nanobeads on the wall. Targeting of nanobeads to the tube was molecularly specific and dependent on, in order of importance, vehicle concentration, wall shear stress, nanobead size, and insonation time. The observation that portions of the microbubble lipid monolayer shell remain attached to adherent nanobeads is important for future consideration of drug transport mechanisms. This versatile method of delivery is shown to enable targeted deposition of nanoparticles in shear flow and could be modified to carry therapeutic agents for controlled release in targeted delivery applications.

A new acoustically-active delivery vehicle was developed by conjugating liposomes and microbubbles, using the high affinity interaction between avidin and biotin. Binding between microbubbles and liposomes each containing 5% DSPE-PEG2kBiotin was highly dependent on avidin concentration and observed above an avidin concentration of 10 nM. With an optimized avidin and liposome concentration, we measured and calculated as high as 1000 to 10,000 liposomes with average diameters of 200 and 100 nm, respectively, attached to each microbubble. Replacing avidin with neutravidin resulted in 3-fold higher binding, approaching the calculated saturation level. Highspeed photography of this new drug delivery vehicle demonstrated that the liposome-bearing microbubbles oscillate in response to an acoustic pulse similar to microbubble contrast agents. Additionally, microbubbles carrying liposomes could be spatially concentrated on a monolayer of PC-3 cells at the focal point of ultrasound beam. As a result of cell-vehicle contact, the liposomes fused with the cells and internalization of NBD-cholesterol occurred shortly after incubation at 37°C, with internalization of NBD-cholesterol substantially enhanced in the acoustic focus.

Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate ␤ 2 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 ϳ75 pM IL-8, corresponding to ligation of only ϳ10 -100 receptors, was sufficient to activate ϳ20,000 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.Neutrophils are among the first cells to respond to acute inflammation through a multistep process of specific bond formation with adhesion molecules up-regulated on the surface of activated endothelium (1, 2). Endothelial and leukocyte selectins function to capture leukocytes from the circulation by rapid bond formation. However, selectins form transient bonds that last a second or less in shear flow and only mediate cell capture and rolling on endothelium (3, 4). Unlike selectins, ␤ 2 integrins (CD18) do not constitutively recognize ligand but require cellular activation to form stable shear-resistant bonds with endothelial ligands including ICAM-1.

Polymorphonuclear leukocyte (PMN) recruitment to vascular endothelium during acute inflammation involves cooperation between selectins, G-proteins, and ␤ 2 -integrins. LFA-1 (CD11a/CD18) affinity correlates with specific adhesion functions because a shift from low to intermediate affinity supports rolling on ICAM-1, whereas high affinity is associated with shear-resistant leukocyte arrest. We imaged PMN adhesion on cytokine-inflamed endothelium in a parallel-plate flow chamber to define the dynamics of ␤ 2 -integrin function during recruitment and transmigration. After arrest on inflamed endothelium, high-affinity LFA-1 aligned along the uropod-pseudopod major axis, which was essential for efficient neutrophil polarization and subsequent transmigration. IntroductionNeutrophils are recruited at vascular sites of acute inflammation by the sequential binding of selectins, CXC chemokines, and ␤ 2 -integrins that function cooperatively to elicit rolling, arrest, and transmigration. From observations of neutrophil recruitment in the murine microcirculation and on endothelial monolayers grown in tissue culture, a number of rules of engagement have emerged. First, Mac-1 (␣ M ␤ 2 ) and LFA-1 (␣ L ␤ 2 ) are necessary and sufficient for neutrophil arrest and transmigration, with each subunit providing distinct adhesive contributions throughout the process from rolling to transmigration. 1-3 Second, polymorphonuclear leukocyte (PMN) rolling on a monolayer of cells coexpressing E-selectin and intercellular adhesion molecule-1 (ICAM-1) is sufficient to induce selectin ligand clustering (PSGL-1 and L-selectin) and to signal a shift in LFA-1 and Mac-1 from low to high affinity to bind ICAM-1. This process is synergistic with chemokine signaling on rolling PMN to amplify the efficiency of arrest. 4,5 LFA-1 appears to function early in this process in that it participates in tethering to ICAM-1 as it shifts from low to intermediate and high affinity. 6,7 How changes in conformation of the heterodimer result in changes in affinity to interact with ICAM-1 and mediate rolling, arrest, and outside-in signaling is only partially defined. 8 Structural studies of LFA-1 reveal that extension and activation of ICAM-1 binding involves an inserted or I-domain on the ␣-subunit and an I-like domain on the ␤-subunit that exerts a pull on the C-terminal ␣ 7 -helix of the ␣-subunit, leading to the open shape of the heterodimer and high-affinity ligand binding. 9,[10][11][12] This conformational shift to high affinity can be stabilized by binding of Mg 2ϩ or Mn 2ϩ or by inside-out signaling by chemokine receptors. 9,13,14 Once activated by a chemokine such as IL-8 or SDF-1, extension and opening of the LFA-1 heterodimer initiate rapid arrest on ICAM-1, as do activated I-domain mutants. 6,[15][16][17][18] Counteracting a shift to high affinity, small molecule allosteric anti-inflammatory inhibitors of LFA-1 function by effectively stabilizing the low-affinity state and antagonize binding to ICAM-1 and leukocyte adhesion. 19,20 XVA143 is one such ...

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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.

An early event in the inflammatory response is neutrophil recruitment to endothelium in response to chemotactic stimulation, which in turn activates CD18-integrin, which anchors neutrophils to the vessel wall under the shear force of blood flow. Activated neutrophils circulating in sickle cell disease (SCD) patients may significantly contribute to vascular occlusions (VOC) as neutrophils adherent to inflamed endothelium recruit sickle red blood cells inducing VOC. To elucidate the mechanisms by which neutrophils may participate in VOC in SCD, CD18-integrin expression and function in fresh blood samples of non-crisis patients were measured by flow cytometry. CD11b/CD18 membrane expression was~70% higher on unstimulated SCD neutrophils than controls, which correlated with a 1-fold higher rate of adhesion to ligand. Unstimulated SCD neutrophils expressed~30,000 active CD18 per cell, while controls expressed~6,000. Stimulation with a low concentration of IL-8 (0.1 nM) upregulated 100% more active CD18 and induced 60% more adhesion of SCD than control neutrophils. These data demonstrate that neutrophils from SCD patients constitutively express active CD18 in blood and respond with enhanced sensitivity to chemokine activation of adhesion, thus increasing their propensity for exuberant adhesion. Am.

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the ␤ 2 integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.Neutrophils circulate in the bloodstream to sites of inflammation where they adhere and transmigrate through the endothelium as the initial step in combating infection and to facilitate wound healing. Recruitment from the circulation involves a multistep process of cell rolling, activation, and arrest. The heterodimeric integrin receptor LFA-1 1 is composed of the ␣L (CD11a) and ␤ 2 (CD18) subunits and is constitutively expressed in a low affinity conformation on the plasma membrane of leukocytes (1-3). Neutrophils encountering chemokines on inflamed endothelium are activated to shift LFA-1 from the low to high affinity conformation, which supports tight binding to endothelial ICAM-1. Increases in integrin affinity correlate in time with adhesion function as recently demonstrated in aggregation of cells expressing ␣4␤1 and vascular cell adhesion molecule (4). ICAM-1 recognizes LFA-1 through an inserted (I) domain in the ␣ subunit. There is strong evidence correlating shifts in I domain conformation to affinity changes in binding ICAM-1. Mutations in I domain residues stabilized distinct structural conformations correlating to LFA-1 affinity. ICAM-1 equilibrium binding constants increase over 4 orders of magnitude ranging between low (i.e. 1600 M), intermediate (i.e. 9 M), and high...