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Special Collection: Molecular Imaging
Seeing the Invisible—Ultrasound Molecular Imaging
Ultrasound molecular imaging has been developed in the past two decades with the goal of non-invasively imaging disease phenotypes on a cellular level not depicted on anatomic imaging. Such techniques already play a role in pre-clinical research for the assessment of disease mechanisms and drug effects, and are thought to in the future contribute to earlier diagnosis of disease, assessment of therapeutic effects and patient-tailored therapy in the clinical field. In this review, we first describe the chemical composition and structure as well as the in vivo behavior of the ultrasound contrast agents that have been developed for molecular imaging.Development of a Translatable Ultrasound Molecular Imaging Agent for Inflammation
This study details the development, characterization and non-clinical efficacy of an ultrasound molecular imaging agent intended for molecular imaging of P-selectin in humans. A targeting ligand based on a recently discovered human selectin ligand was manufactured as fusion protein, and activity for human and mouse P- and E-selectin was evaluated by functional immunoassay. The targeting ligand was covalently conjugated to a lipophilic anchor inserted into a phospholipid microbubble shell. Three lots of the targeted microbubble drug product, TS-07-009, were produced, and assays for size distribution, zeta potential and morphology were established.Surface Modification with Lactadherin Augments the Attachment of Sonazoid Microbubbles to Glycoprotein IIb/IIIa
Arginine–glycine–aspartate (RGD)-carrying microbubbles (MBs) have been utilized as a specific contrast agent for glycoprotein IIb/IIIa (αIIbβ3 integrin)-expressing activated platelets in ultrasound molecular imaging. Recently, we found that surface modification with lactadherin provides the RGD motif on the surface of phosphatidylserine-containing clinically available MBs, Sonazoid. Here, we examined the potential of lactadherin-bearing Sonazoid MBs to be targeted MBs for glycoprotein IIb/IIIa using the custom-designed in vitro settings with recombinant αIIbβ3 integrin, activated platelets or erythrocyte-rich human clots.Ultrasound Molecular Imaging of Atherosclerosis Using Small-Peptide Targeting Ligands Against Endothelial Markers of Inflammation and Oxidative Stress
The aim of this study was to evaluate a panel of endothelium-targeted microbubble (MB) ultrasound contrast agents bearing small peptide ligands as a human-ready approach for molecular imaging of markers of high-risk atherosclerotic plaque. Small peptide ligands with established affinity for human P-selectin, VCAM-1, LOX-1 and von Willebrand factor (VWF) were conjugated to the surface of lipid-stabilized MBs. Contrast-enhanced ultrasound (CEUS) molecular imaging of the thoracic aorta was performed in wild-type and gene-targeted mice with advanced atherosclerosis (DKO).Quantification of Endothelial αvβ3 Expression with High-Frequency Ultrasound and Targeted Microbubbles: In Vitro and In Vivo Studies
Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques. The precursory molecular or cellular pro-angiogenic events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. This study was designed to investigate the feasibility of ultrasound molecular imaging of endothelial αvβ3 expression in vitro and in vivo using αvβ3-targeted ultrasound contrast agents (UCAs).Molecular Acoustic Angiography: A New Technique for High-resolution Superharmonic Ultrasound Molecular Imaging
Ultrasound molecular imaging utilizes targeted microbubbles to bind to vascular targets such as integrins, selectins and other extracellular binding domains. After binding, these microbubbles are typically imaged using low pressures and multi-pulse imaging sequences. In this article, we present an alternative approach for molecular imaging using ultrasound that relies on superharmonic signals produced by microbubble contrast agents. Bound bubbles were insonified near resonance using a low frequency (4 MHz) element and superharmonic echoes were received at high frequencies (25–30 MHz).VEGFR2-Targeted Contrast-Enhanced Ultrasound to Distinguish between Two Anti-Angiogenic Treatments
The aim of this study was to evaluate the capacity of BR55, an ultrasound contrast agent specifically targeting vascular endothelial growth factor receptor 2 (VEGFR2), to distinguish the specific anti-VEGFR2 therapy effect of sunitinib from other anti-angiogenic effects of a therapy (imatinib) that does not directly inhibit VEGFR2. Sunitinib, imatinib and placebo were administered daily for 11 d (264 h) to 45 BalbC mice bearing ectopic CT26 murine colorectal carcinomas. During the course of therapy, B-mode ultrasound, contrast-enhanced ultrasound and VEGFR2-targeted contrast-enhanced ultrasound were performed to assess tumor morphology, vascularization and VEGFR2 expression, respectively.Nitric Oxide-Enhanced Molecular Imaging of Atheroma using Vascular Cellular Adhesion Molecule 1-Targeted Echogenic Immunoliposomes
The aim of this study was to determine whether pre-treatment with nitric oxide-loaded echogenic liposomes (NO-ELIP) plus ultrasound can improve highlighting by molecularly targeted (anti-vascular cell adhesion molecule 1 [VCAM-1]) ELIP of atheroma components. Atherosclerotic animals were treated with anti-VCAM-1-ELIP or immunoglobulin (IgG)-ELIP. Each group was selected at random to receive pre-treatment with standard ELIP plus ultrasound, NO-ELIP without ultrasound and NO-ELIP plus ultrasound. Intravascular ultrasound highlighting data for the same arterial segments were collected before and after treatment.Influence of Repetitive Contrast Agent Injections on Functional and Molecular Ultrasound Measurements
Quantitative contrast-enhanced ultrasound plays an important role in tumor characterization and treatment assessment. Besides established functional ultrasound techniques, ultrasound molecular imaging using microbubbles targeted to disease-associated markers is increasingly being applied in pre-clinical studies. Often, repeated injections of non-targeted or targeted microbubbles during the same imaging session are administered. However, the influence of repeated injections on the accuracy of the quantitative data is unclear.VEGFR2-Targeted Molecular Imaging in the Mouse Embryo: An Alternative to the Tumor Model
As a tumor surrogate, the mouse embryo presents as an excellent alternative for examining the binding of angiogenesis-targeting microbubbles and assessing the quantitative nature of molecular ultrasound. We establish the validity of this model by developing a robust method to study microbubble kinetic behavior and investigate the reproducibility of targeted binding in the murine embryo. Vascular endothelial growth factor receptor 2 (VEGFR2)-targeted (MBV), rat immunoglobulin G2 (IgG2) control antibody-targeted (MBC) and untargeted (MBU) microbubbles were introduced into vasculature of living mouse embryos.An Animal Model Allowing Controlled Receptor Expression for Molecular Ultrasound Imaging
Reported in this study is an animal model system for evaluating targeted ultrasound (US) contrast agents binding using adenoviral (Ad) vectors to modulate cellular receptor expression. An Ad vector encoding an extracellular hemagglutinin (HA) epitope tag and a green fluorescent protein (GFP) reporter was used to regulate receptor expression. A low and high receptor density (in breast cancer tumor bearing mice) was achieved by varying the Ad dose with a low plaque forming unit (PFU) on day 1 and high PFU on day 2 of experimentation.Effects of Acoustic Radiation Force on the Binding Efficiency of BR55, a VEGFR2-Specific Ultrasound Contrast Agent
This work describes an in vivo study analyzing the effect of acoustic radiation force (ARF) on the binding of BR55 VEGFR2-specific contrast-agent microbubbles in a model of prostatic adenocarcinoma in rat. A commercial ultrasound system was modified by implementing high duty-cycle 3.5-MHz center frequency ARF bursts in a scanning configuration. This enabled comparing the effects of ARF on binding in tumor and healthy tissue effectively in the same field of view. Bubble binding was established by measuring late-phase enhancement in amplitude modulation (AM) contrast-specific imaging mode (4 MHz, 150 kPa) 10 min after agent injection when the unbound bubbles were cleared from the circulation.Molecular Imaging With Targeted Perfluorocarbon Nanoparticles: Quantification of the Concentration Dependence of Contrast Enhancement for Binding to Sparse Cellular Epitopes
Targeted, liquid perfluorocarbon nanoparticles are effective agents for acoustic contrast enhancement of abundant cellular epitopes (e.g., fibrin in thrombi) and for lower prevalence binding sites, such as integrins associated with tumor neovasculature. In this study, we sought to delineate the quantitative relationship between the extent of contrast enhancement of targeted surfaces and the density (and concentration) of bound perfluorocarbon (PFC) nanoparticles. Two dramatically different substrates were utilized for targeting.
