Rationale and Objectives-Ultrasound molecular imaging is an emerging technique for sensitive detection of intravascular targets. Molecular imaging of angiogenesis has strong potential for both clinical use and as a research tool in tumor biology and the development of antiangiogenic therapies. Our objective is to develop a robust microbubble (MB) ultrasound contrast agent platform to which targeting ligands can be conjugated by biocompatible, covalent conjugation chemistry, and to develop a pure low mechanical index imaging processing method and corresponding quantifying method. The microbubbles and the imaging methods were evaluated in a mouse model of breast cancer in vivo.Materials and Methods-We utilized a cyclic RGD (cRGD) pentapeptide containing a terminal cysteine group conjugated to the surface of MB bearing pyridyldithio-propionate (PDP) for targeting α v β 3 integrins. As negative controls, MB without a ligand or MB bearing a scrambled sequence (cRAD) were prepared. To enable characterization of peptides bound to MB surfaces, the cRGD peptide was labeled with FITC and detected by plate fluorometry, flow cytometry, and fluorescence microscopy. Targeted adhesion of cRGD-MB was demonstrated in an in vitro flow adhesion assay against recombinant murine α v β 3 integrin protein and α v β 3 integrin-expressing endothelial cells (bEnd.3). The specificity of cRGD-MB for α v β 3 integrin was demonstrated by treating bEnd.3 EC with a blocking antibody. A murine model of mammary carcinoma was used to assess targeted adhesion and ultrasound molecular imaging in vivo. The targeted microbubbles were visualized using a low mechanical index contrast imaging pulse sequence, and quantified by intensity normalization and two-dimensional Fourier transform analysis, Results-The cRGD ligand concentration on the MB surface was ~8.2 × 10 6 molecules/MB. At a wall shear stress of 1.0 dynes/cm 2 , cRGD-MB exhibited 5-fold higher adhesion to immobilized recombinant α v β 3 integrin relative to non-targeted MB and cRAD-MB controls. Similarly, cRGD-MB showed significantly greater adhesion to bEnd.3 EC compared to non-targeted MB and cRAD-MB. In addition, cRGD-MB, but not non-targeted MB or cRAD-MB, showed significantly enhanced contrast signals with a high tumor-to-background ratio. bEnd.3 was reduced by 80% after using anti-α v monoclonal antibody to treat bEnd.3. The normalized image intensity amplitude was ~0.8 seven minutes after the administration of cRGD-MB relative to the intensity amplitude at the time of injection, while the spatial variance in image intensity improved the detection of bound agents. The accumulation of cRGD-MB was blocked by pre-administration with an anti-α v blocking antibody.Conclusion-The results demonstrate the functionality of a novel microbubble contrast agent covalently coupled to an RGD peptide for ultrasound molecular imaging of α v β 3 integrin and the feasibility of quantitative molecular ultrasound imaging with a low mechanical index.

The objective of the study was to examine the role of acoustic power intensity and microbubble and plasmid concentrations on transfection efficiency in HEK-293 cells using a sonoporator with a 1-MHz transducer. A green fluorescent protein (GFP) reporter plasmid was delivered in as much as 80% of treated cells, and expression of the GFP protein was observed in as much as 75% of cells, using a power intensity of 2 W/cm 2 with a 25% duty cycle. In addition, the relative transfection abilities of a lipid noncationic and cationic microbubble platform were investigated. As a positive control, cells were transfected using Lipofectamine reagent. Cell survival and transfection efficiency were inversely proportional to acoustic power and microbubble concentration. Our results further demonstrated that high-efficiency transfection could be achieved, but at the expense of cell loss. Moreover, direct conjugation of plasmid to the microbubble did not appear to significantly enhance transfection efficiency under the examined conditions, although this strategy may be important for targeted transfection in vivo.

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Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract (GI) for which treatments with immunosuppressive drugs have significant side-effects. Consequently, there is a clinical need for site-specific and non-toxic delivery of therapeutic genes or drugs for CD and related disorders such as inflammatory bowel disease. The aim of this study was to validate a gene delivery platform based on ultrasound-activated lipid-shelled microbubbles (MBs) targeted to inflamed mesenteric endothelium in the CD-like TNFΔARE mouse model. MBs bearing luciferase plasmid were functionalized with antibodies to MAdCAM-1 (MB-M) or VCAM-1 (MB-V), biomarkers of gut endothelial cell inflammation and evaluated in an in vitro flow chamber assay with appropriate ligands to confirm targeting specificity. Following MB retro-orbital injection in TNFΔARE mice, the mean contrast intensity in the ileocecal region from accumulated MB-M and MB-V was 8.5-fold and 3.6-fold greater, respectively, compared to MB-C. Delivery of luciferase plasmid to the GI tract in TNFΔARE mice was achieved by insonating the endothelial cell-bound agents using a commercial sonoporator. Luciferase expression in the midgut was detected 48 h later by bioluminescence imaging and further confirmed by immunohistochemical staining. The liver, spleen, heart, and kidney had no detectable bioluminescence following insonation. Transfection of the microcirculation guided by a targeted, acoustically-activated platform such as an ultrasound contrast agent microbubble has the potential to be a minimally-invasive treatment strategy to ameliorate CD and other inflammatory conditions.

Background & Aims Lymphocyte recruitment to sites of inflammation requires the sequential engagement of adhesion molecules and chemokine receptors. In the current studies we analyzed the role of CD44 for the development of chronic small-intestinal inflammatory infiltrates in vivo. Methods By using a tumor necrosis factor (TNF)-driven model of chronic ileitis (ie, B6.129P-TNFΔAU-rich element [ARE]) that recapitulates many features of Crohn’s disease, we noticed dynamic changes in the expression and functional state of CD44 and its ligand hyaluronan via enzyme-linked immunosorbent assay, real-time reverse-transcription polymerase chain reaction, immunohistochemistry, and flow cytometry. In addition, we assessed the role of lymphocyte populations during induction of ileitis through adoptive transfer studies, and generated CD44-deficient TNFΔARE mice to assess the role of CD44 for development of ileitis. Results Soluble hyaluronan levels and expression of hyaluronan synthase-1 were increased in TNFΔARE mice. This coincided with increased expression of CD44 (including variant 7) and reactivity towards hyaluronan on CD4+ T cells. CD44 was spatially co-localized with the gut-homing integrin α4β7, spatially linking lymphocyte rolling with arrest. These cells had an effector phenotype because they lacked L-selectin and a higher proportion in diseased mice produced TNF and interleukin-2 compared with wild-type littermates. Lastly, CD4+ but not CD8+ T cells conferred ileitis to RAG−/− recipients and deficiency of one or both alleles of the CD44 gene resulted in attenuation of the severity of ileitis in TNFΔARE mice. Conclusions Our findings support an important role of CD44 expressed by CD4+ and CD8+ for development of ileitis mediated by TNF overproduction.