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WIREs Nanomed Nanobiotechnol
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Stimulus–responsive ultrasound contrast agents for clinical imaging: motivations, demonstrations, and future directions

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Microbubble ultrasound contrast agents allow imaging of the vasculature with excellent resolution and signal‐to‐noise ratios. Contrast in microbubbles derives from their interaction with an ultrasound wave to generate signal at harmonic frequencies of the stimulating pulse; subtracting the elastic echo caused by the surrounding tissue can enhance the specificity of these harmonic signals significantly. The nonlinear acoustic emission is caused by pressure‐driven microbubble size fluctuations, which in both theoretical descriptions and empirical measurements was found to depend on the mechanical properties of the shell that encapsulates the microbubble as well as stabilizes it against the surrounding aqueous environment. Thus biochemically induced switching between a rigid ‘off’ state and a flexible ‘on’ state provides a mechanism for sensing chemical markers for disease. In our research, we coupled DNA oligonucleotides to a stabilizing lipid monolayer to modulate stiffness of the shell and thereby induce stimulus–responsive behavior. In initial proof‐of‐principle studies, it was found that signal modulation came primarily from DNA crosslinks preventing the microbubble size oscillations rather than merely damping the signal. Next, these microbubbles were redesigned to include an aptamer sequence in the crosslinking strand, which not only allowed the sensing of the clotting enzyme thrombin but also provided a general strategy for sensing other soluble biomarkers in the bloodstream. Finally, the thrombin‐sensitive microbubbles were validated in a rabbit model, presenting the first example of an ultrasound contrast agent that could differentiate between active and inactive clots for the diagnosis of deep venous thrombosis. WIREs Nanomed Nanobiotechnol 2015, 7:111–123. doi: 10.1002/wnan.1285 This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Biology-Inspired Nanomaterials > Lipid-Based Structures Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures
Microbubble design and synthesis. (a) Schematic of crosslinking and reactivation in a microbubble shell. (b) Synthesis of conjugates of 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphoethanolamine, poly(acrylic acid), and A15 oligonucleotides. (c) Bright field optical micrograph of synthesized microbubbles. Scale bar (yellow) = 5 µm. (d, e) 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine and 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphatic acid form the dominant portion of the shell. (Reprinted with permission from Ref . Copyright 2011 Wiley)
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In vivo imaging of deep venous thrombosis: activation of (a) thrombin‐sensitive and (b) thrombin‐insensitive crosslinked microbubbles at clot site in cadence mode. Arrows point at probable clot site as determined by physical tie. Blue outlines indicate vena cava walls. (c) Acoustic signal generated within the clot site over time for thrombin‐sensitive (red) or thrombin‐insensitive (blue) crosslinked microbubbles within the inferior vena cava. (d) Acoustic signal generated as bubble encounter the clot and become active. This corresponds to area of interest highlighted in blue. (Reprinted with permission from Ref . Copyright 2013 Elsevier)
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(a) Ultrasound signal retained over time in citrate‐treated nonclotting blood. Purple circle = 0 mg/mL PAA‐DNA, 0.3 mg/mL PEG, blue diamond = 0.3 mg/mL PAA‐DNA, 0 mg/mL PEG, red square = 0.3 mg/mL PAA‐DNA, 0.075 mg/mL PEG, green triangle = 0.3 mg/mL PAA‐DNA, 0.225 mg/mL PEG. (b, c) Aptamer‐crosslinked microbubbles when a clot is absent (b) or present (c). (d) Mean signal generated by thrombin‐sensitive microbubbles within the clot site in the presence (red) or absence (blue) of thrombin over time. T = 0 s corresponds to when blood first begins to circulate. (Reprinted with permission from Ref . Copyright 2013 Elsevier)
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Sequences of crosslinking strand (top) and microbubble‐bound strand (bottom). The aptamer sequence is denoted in red, while the portion of the bound strand complementary to the aptamer is blue. (a) Freshly drawn rabbit blood clots in a few min (b–d) Contrast‐enhanced ultrasound sonograms of (b) aptamer‐crosslinked microbubbles, (c) non‐aptamer‐crosslinked microbubbles, and (d) PEG‐coated, freely oscillating microbubbles in freshly drawn rabbit blood, 5 min after mixing. (e) Frequency response of microbubbles as function of thrombin addition at 2.25 MHz. At higher thrombin loadings the second harmonic at 4.5 MHz appears as a sharp peak, while at moderate amounts the response has a large broadband contribution. (f) Integrations of data from (e) showing that total power output is approximately equal at thrombin concentrations ≥50 nM. (Reprinted with permission from Ref . Copyright 2012 Wiley)
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Ultrasound analysis of DNA‐crosslinkable microbubbles. (a) Plotting microbubble onset pressure versus inverse microbubble radius yields a linear fit for uncrosslinked (green) and crosslinked (red) microbubbles. The intercept corresponds to an activation pressure of approximately 300 kPa. (b) Integrated light scattering signal for oscillating uncrosslinked (green) and crosslinked (red) microbubbles. While the size fluctuations of uncrosslinked bubbles appear to increase steadily with increasing incident pressure, the crosslinked bubbles lose signal as pressure increases. (c) Contrast‐enhanced ultrasound image of crosslinked microbubbles in phantom. (d) Contrast‐enhanced ultrasound image of same suspension after addition of A30. (Reprinted with permission from Ref . Copyright 2011 Wiley)
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Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures
Biology-Inspired Nanomaterials > Lipid-Based Structures
Diagnostic Tools > In Vivo Nanodiagnostics and Imaging

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