Ultrasound Molecular Imaging Research Articles

Rapid Copper-free Click Conjugation to Lipid-Shelled Microbubbles for Ultrasound Molecular Imaging of Murine Bowel Inflammation.

Microbubbles are ultrasound contrast agents that can adhere to disease-related vascular biomarkers when functionalized with binding ligands such as antibodies or peptides. The biotin-streptavidin approach has predominantly been used as the microbubble labeling approach in preclinical imaging. However, due to the immunogenicity of avidin in humans, it is not suitable for clinical translation. What would aid clinical translation is a simple and effective microbubble functionalization approach that could be directly translated from animals to humans. We developed a targeted microbubble to P-selectin, a vascular inflammatory marker, labeled using a strain-promoted [3 + 2] azide-alkyne (azide-DBCO) reaction, comparing its ability to detect bowel inflammation to that of P-selectin targeted microbubbles labeled with a traditional biotin-streptavidin approach. Bowel inflammation was chemically induced using 2,4,6-trinitrobenzenesulfonic acid (TNBS) in Balb/C mice. Each mouse received both non-targeted and P-selectin targeted microbubbles (either biotin-streptavidin or azide-DBCO). Using the biotin-streptavidin reaction, there was a significant increase in the ultrasound molecular imaging signal in inflamed mice using P-selectin targeted (2.30 ± 0.91 a.u.) compared to isotype control microbubbles (1.14 ± 0.7 a.u.) (p = 0.009). Using the azide-DBCO reaction, there was a similar increase in the ultrasound molecular imaging signal in inflamed mice (2.54 ± 0.56 a.u) compared to the isotype control (0.44 ± 0.25 a.u) (p = 0.009). There were no significant differences between the two labeling approaches between non-targeted and P-selectin targeted microbubbles. Mouse inflammatory phenotypes and expression of P-selectin were validated using histology and immunostaining. We constructed P-selectin targeted microbubbles using an azide-DBCO click reaction, which could detect bowel inflammation in vivo. This reaction generated a similar ultrasound molecular imaging signal to biotin-strepavidin-labeled microbubbles. These data show the potential of click chemistry conjugation (azide-DBCO) as a quick, cost-efficient, and clinically translatable approach for developing targeted microbubbles.

Effect of microbubble and acoustic radiation force parameters on αvβ3–microbubble targeting

Ultrasound molecular imaging (USMI) employs targeted microbubbles (MBs) decorated with specific ligands to enhance the local visualization of vascular structures. It has been demonstrated that cyclo Arg-Gly-Asp (cRGD) targeted MBs in combination with acoustic radiation force (ARF) can bind to αvβ3 integrins providing enhanced images of the tumor vasculature. Therefore, the goal of this work was to study and elucidate the role of ARF and MB parameters on the targeting of cRGD-MBs using an in vitro αvβ3-coated microvessel phantom. Labeled polydisperse and 2-μm size- isolated MBs decorated with cRGD with buried-ligand architecture (BLA) were pumped at 1 ml/h in an αvβ3-coated microvessel (D = 200 μm) and pushed using ARF. At higher monodisperse MB concentrations, the specific attachment follows a linear behavior independent of the frequency applied. However, at lower MB concentrations, the attachment becomes linearly dependent on the frequency. Finally, attachment of monodisperse population of RGD-MBs increased 8- and 3- fold than polydisperse MBs. In conclusion, we elucidated that the use of a monodisperse population of MBs improves the molecular specificity for αvβ3 integrins.

Ultrasound contrast agents: From buckling dynamics to swimming

Cleverly engineered microswimmers have been of increasing scientific interest, as they show great promise in various biomedical applications. In this study, we propose a novel mechanism of propulsion in fluids at the microscale, using a buckling mechanism activated by pressure waves. We considered an in vivo-friendly hollow elastic shell of micrometric size composed of a lipidic membrane enclosing a gas bubble. Such microshells are approved for clinical use as diagnostic ultrasound contrast agents (UCAs). We experimentally investigate the buckling dynamics of microshells upon an increase of external pressure. The effect of the driving frequency is studied as well as that of the size and mechanical properties of the microshells. We evidence a non-zero displacement upon a complete cycle of deflation and re-inflation of the microshells, which includes buckling events. The proposed propulsion mechanism whose direction is controlled in the shell reference frame can be an answer to the problem of directivity accounted in the acoustic radiation force technique used in ultrasound molecular imaging and drug delivery.

The spatial distribution of the ligand on targeted microbubbles influences the binding efficacy

An important trait of targeted microbubbles is their binding to biomarkers for ultrasound molecular imaging and drug delivery. Using organic solvents in phospholipid-coated microbubble production results in a homogeneous ligand distribution, while dispersion of lipids directly into aqueous medium results in a heterogeneous ligand distribution. In this study, we compared the binding efficacy of ανβ3-targeted microbubbles with a homogeneous or heterogeneous ligand distribution in vitro and in vivo using confocal microscopy. For in vitro studies, human umbilical vein endothelial cells grown statically and under physiological flow were used. For in vivo studies, chicken embryos (day 5) were used. Microbubbles having a homogeneous ligand distribution bound 1.55× more than microbubbles having a heterogeneous ligand distribution in vitro statically, while in vitro under flow this was 1.49× more at 1.25 dyn/cm2 and 1.56× more at 2.22 dyn/cm2; in vivo this was 1.25× more. The dissociation rate in vitro was lower for bound microbubbles with a homogeneous than heterogeneous ligand distribution at low shear stresses (1–5 dyn/cm2). In conclusion, when producing phospholipid-coated targeted microbubbles using organic solvents is preferable over directly dispersing phospholipids in aqueous medium for optimal binding. [Funding by the Phospholipid Research Center (No. KKO-2017-057/1-1) and NWO (VIDI Project No.17543) is gratefully acknowledged.]

Spatiotemporal decomposition methods for nanobubble contrast-enhanced ultrasound

Nanobubble ultrasound contrast agents (NBs) offer enhanced access to deep tumor tissue by extrapolation from vasculature. When combined with targeting moieties, shell-stabilized nanobubbles can outperform other agents used in molecular ultrasound imaging due to their small size. The visualization and decoupling of extravasated and intravascular NBs is critical to furthering nanobubble molecular imaging with ultrasound. Spatiotemporal processing of echo data, usually via singular value decomposition (SVD), enables enhanced decoupling of signals of stationary from moving scatterers and therefore provides a novel tool for analysis of NB extravasation into the parenchyma. Instead of trivial solutions, we have developed non-negative SVD (nnSVD) methods applicable to most commercial scanners without requiring the raw data. Additionally, our formulation allows addition of proper regularizers, such as sparsity, to further expand the decomposition dimensions. We validate these methods in phantom models and present how the suggested decompositions can distinctly decouple flowing from extravasated NBs in in vivo tumour models with enhanced permeability. Our results show the successful tracking of nanobubble accumulation in tumour tissue and can serve as a relative metric for molecular targeting of prostate cancer. Further exploration of these methods could be used to improve long-term imaging and drug delivery capabilities of NBs.

Self-Enhanced Acoustic Impedance Difference Strategy for Detecting the Acidic Tumor Microenvironment.

B-mode ultrasound imaging is a significant anatomic technique in clinic, which can display the anatomic variation in tissues. However, it is difficult to evaluate the functional state of organs and display the physiological information in organisms such as the tumor acidic microenvironment (TME). Herein, inspired by the phenomenon of sonographic acoustic shadow during detecting calculus in clinic, a strategy of self-enhanced acoustic impedance difference is proposed to monitor the acidic TME. BiF3@PDA@PEG (BPP) nanoparticles can self-aggregate in a specific response to the acidic TME to form huge "stones" BiF3@PDA, resulting in an increase of local tumor density, and further causing a significant acoustic impedance difference. In in vitro experiments, the enhanced ultrasound signals change from 15.2 to 196.4 dB, which can discriminate different pH values from 7.0 to 5.0, and the sensitivity can reach to 0.2 value. In in vivo experiments, the enhanced ultrasound signal is 107.7 dB after BPP self-aggregated, displaying the weak acidic TME that has a close relationship with the size and species of the tumor. More importantly, the accuracy is away from the interference of pressure because huge "stones" BiF3@PDA change little. However, SonoVue microbubbles will diffuse and rupture under pressure, which results in false positive signals. To sum up, this strategy will be helpful to the further development of ultrasound molecular imaging.

Expanding the Role of Ultrasound for the Characterization of Renal Masses.

: BackgroundThe incidental detection of renal masses has been steadily rising. As a significant proportion of renal masses that are surgically treated are benign or indolent in nature, there is a clear need for better presurgical characterization of renal masses to minimize unnecessary harm. Ultrasound is a widely available and relatively inexpensive real-time imaging technique, and novel ultrasound-based applications can potentially aid in the non-invasive characterization of renal masses. Evidence acquisition: We performed a narrative review on novel ultrasound-based techniques that can aid in the non-invasive characterization of renal masses. Evidence synthesis: Contrast-enhanced ultrasound (CEUS) adds significant diagnostic value, particularly for cystic renal masses, by improving the characterization of fine septations and small nodules, with a sensitivity and specificity comparable to magnetic resonance imaging (MRI). Additionally, the performance of CEUS for the classification of benign versus malignant renal masses is comparable to that of computed tomography (CT) and MRI, although the imaging features of different tumor subtypes overlap significantly. Ultrasound molecular imaging with targeted contrast agents is being investigated in preclinical research as an addition to CEUS. Elastography for the assessment of tissue stiffness and micro-Doppler imaging for the improved detection of intratumoral blood flow without the need for contrast are both being investigated for the characterization of renal masses, though few studies have been conducted and validation is lacking. Conclusions: Several novel ultrasound-based techniques have been investigated for the non-invasive characterization of renal masses. CEUS has several advantages over traditional grayscale ultrasound, including the improved characterization of cystic renal masses and the potential to differentiate benign from malignant renal masses to some extent. Ultrasound molecular imaging offers promise for serial disease monitoring and the longitudinal assessment of treatment response, though this remains in the preclinical stages of development. While elastography and emerging micro-Doppler techniques have shown some encouraging applications, they are currently not ready for widespread clinical use.

Combined legumain- and integrin-targeted nanobubbles for molecular ultrasound imaging of breast cancer

Molecular ultrasound imaging is a promising strategy for non-invasive and precise cancer diagnosis. Previously reported ultrasound contrast agents (UCAs) are mostly microbubbles or nanobubbles (NBs) larger than 200 nm, leading to less efficient tumor delivery. Here we synthesized NBs with a small size (~49 nm) and modified the NB surface with alanine-alanine-asparagine (NB-A) or arginine-glycine-aspartic acid peptide (NB-R) for concurrent active targeting towards legumain in tumor cells and integrin in tumor neovasculature. In vitro, the NB-A and NB-R presented echogenicity comparable with SonoVue MBs and showed specific binding with tumors cells and endothelial cells, respectively. In vivo, the combined NB-A/NB-R accumulated in tumor tissues selectively and provided ultrasound signals with prolonged duration and that were significantly stronger than non-targeted NBs, single-targeted NBs and SonoVue MBs. Overall, the dual targeted NBs served as efficient UCAs for specific imaging of breast cancer, and hold great potential for general cancer diagnosis/monitoring in the future.

Displacement Patterns in Magnetomotive Ultrasound Explored by Finite Element Analysis.

Magnetomotive ultrasound is an emerging technique that enables detection of magnetic nanoparticles. This has implications for ultrasound molecular imaging, and potentially addresses clinical needs regarding determination of metastatic infiltration of the lymphatic system. Contrast is achieved by a time-varying magnetic field that sets nanoparticle-laden regions in motion. This motion is governed by vector-valued mechanical and magnetic forces. Understanding how these forces contribute to observed displacement patterns is important for the interpretation of magnetomotive ultrasound images. Previous studies have captured motion adjacent to nanoparticle-laden regions that was attributed to diamagnetism. While diamagnetism could give rise to a force, it cannot fully account for the observed displacements in magnetomotive ultrasound. To isolate explanatory variables of the observed displacements, a finite element model is set up. Using this model, we explore potential causes of the unexplained motion by comparing numerical models with earlier experimental findings. The simulations reveal motion outside particle-laden regions that could be attributed to mechanical coupling and the principle of mass conservation. These factors produced a motion that counterbalanced the time-varying magnetic excitation, and whose extent and distribution was affected by boundary conditions as well as compressibility and stiffness of the surroundings. Our findings emphasize the importance of accounting for the vector-valued magnetic force in magnetomotive ultrasound imaging. In an axisymmetric geometry, that force can be represented by a simple scalar expression, an oversimplification that rapidly becomes inaccurate with distance from the symmetry axis. Additionally, it results in an underestimation of the vertical force component by up to 30%. We therefore recommend using the full vector-valued force to capture the magnetic interaction. This study enhances our understanding of how forces govern magnetic nanoparticle displacement in tissue, contributing to accurate analysis and interpretation of magnetomotive ultrasound imaging.

Research Progress of Targeted Ultrasound Contrast Agent BR55

BR55 is an ultrasound contrast agent targeting vascular endothelial growth factor receptor 2,which can be used to detect tumor neovascularization and improve the diagnostic accuracy.Overseas researchers have used BR55 for human ultrasound molecular imaging,which showed good safety and tolerance.We reviewed the research progress on BR55 applied in the evaluation of tumor neovascularization from the composition,characteristics,animal experiments,and clinical studies of BR55.

Theranostic Microbubbles with Homogeneous Ligand Distribution for Higher Binding Efficacy.

Phospholipid-coated targeted microbubbles are used for ultrasound molecular imaging and locally enhanced drug delivery, with the binding efficacy being an important trait. The use of organic solvent in microbubble production makes the difference between a heterogeneous or homogeneous ligand distribution. This study demonstrates the effect of ligand distribution on the binding efficacy of phospholipid-coated ανβ3-targeted microbubbles in vitro using a monolayer of human umbilical-vein endothelial cells and in vivo using chicken embryos. Microbubbles with a homogeneous ligand distribution had a higher binding efficacy than those with a heterogeneous ligand distribution both in vitro and in vivo. In vitro, 1.55× more microbubbles with a homogeneous ligand distribution bound under static conditions, while this was 1.49× more under flow with 1.25 dyn/cm2, 1.56× more under flow with 2.22 dyn/cm2, and 1.25× more in vivo. The in vitro dissociation rate of bound microbubbles with homogeneous ligand distribution was lower at low shear stresses (1–5 dyn/cm2). The internalized depth of bound microbubbles was influenced by microbubble size, not by ligand distribution. In conclusion, for optimal binding the use of organic solvent in targeted microbubble production is preferable over directly dispersing phospholipids in aqueous medium.

Contrast Enhanced Ultrasound Molecular Imaging of Spontaneous Chronic Inflammatory Bowel Disease in an Interleukin-2 Receptor α-/- Transgenic Mouse Model Using Targeted Microbubbles.

Inflammatory bowel disease (IBD) is a lifelong inflammatory disorder with relapsing–remission cycles, which is currently diagnosed by clinical symptoms and signs, along with laboratory and imaging findings. However, such clinical findings are not parallel to the disease activity of IBD and are difficult to use in treatment monitoring. Therefore, non-invasive quantitative imaging tools are required for the multiple follow-up exams of IBD patients in order to monitor the disease activity and determine treatment regimens. In this study, we evaluated a dual P- and E-selectin-targeted microbubble (MBSelectin) in an interleukin-2 receptor α deficient (IL-2Rα−/−) spontaneous chronic IBD mouse model for assessing long-term anti-inflammatory effects with ultrasound molecular imaging (USMI). We used IL-2Rα−/− (male and female on a C57BL/6 genetic background; n = 39) and C57BL/6 wild-type (negative control; n = 6) mice for the study. USMI of the proximal, middle, and distal colon was performed with MBSelectin using a small animal scanner (Vevo 2100) up to six times in each IL-2Rα−/− mouse between 6–30 weeks of age. USMI signals were compared between IL-2Rα−/− vs. wild-type mice, and sexes in three colonic locations. Imaged colon segments were analyzed ex vivo for inflammatory changes on H&E-stained sections and for selectin expression by immunofluorescence staining. We successfully detected spontaneous chronic colitis in IL-2Rα−/− mice between 6–30 weeks (onset at 6–14 weeks) compared to wild-type mice. Both male and female IL-2Rα−/− mice were equally (p = 0.996) affected with the disease, and there was no significant (p > 0.05) difference in USMI signals of colitis between the proximal, middle, and distal colon. We observed the fluctuating USMI signals in IL-2Rα−/− mice between 6–30 weeks, which might suggest a resemblance of the remission-flare pattern of human IBD. The ex vivo H&E and immunostaining further confirmed the inflammatory changes, and the high expression of P- and E-selectin in the colon. The results of this study highlight the IL-2Rα−/− mice as a chronic colitis model and are suitable for the long-term assessment of treatment response using a dual P- and E-selectin-targeted USMI.

Acoustic Molecular Imaging Beyond the Diffraction Limit In Vivo.

Ultrasound molecular imaging (USMI) is a technique used to noninvasively estimate the distribution of molecular markers in vivo by imaging microbubble contrast agents (MCAs) that have been modified to target receptors of interest on the vascular endothelium. USMI is especially relevant for preclinical and clinical cancer research and has been used to predict tumor malignancy and response to treatment. In the last decade, methods that improve the resolution of contrast-enhanced ultrasound by an order of magnitude and allow researchers to noninvasively image individual capillaries have emerged. However, these approaches do not translate directly to molecular imaging. In this work, we demonstrate super-resolution visualization of biomarker expression in vivo using superharmonic ultrasound imaging (SpHI) with dual-frequency transducers, targeted contrast agents, and localization microscopy processing. We validate and optimize the proposed method in vitro using concurrent optical and ultrasound microscopy and a microvessel phantom. With the same technique, we perform a proof-of-concept experiment in vivo in a rat fibrosarcoma model and create maps of biomarker expression co-registered with images of microvasculature. From these images, we measure a resolution of 23 μm, a nearly fivefold improvement in resolution compared to previous diffraction-limited molecular imaging studies.

Contrast-Enhanced Ultrasound Molecular Imaging in Atherosclerosis Research.

The management of cardiovascular conditions will likely be improved by noninvasive in vivo molecular imaging technologies that can provide earlier or more accurate diagnosis. These techniques are already having a positive impact in preclinical research by providing insight into disease pathobiology or efficacy of new therapies. Contrast enhanced ultrasound (CEU) molecular imaging is a technique that relies on the ultrasound detection of targeted microbubble contrast agents to examine molecular or cellular events that occur at the blood pool-endothelial interface. For the most part, targeted contrast agents are composed of encapsulated gas microbubbles (MBs) that are 2-4μm in diameter, or other acoustically active micro- or nanoparticles. These agents bear several tens of thousands of binding molecules per particle. Because nonadhered agent is cleared rapidly, CEU molecular imaging can be performed in a matter of minutes. MBs are detected using contrast-specific techniques that generate and receive nonlinear signals produced by MB cavitation, thereby increasing signal-to-noise ratio. Dedicated kinetic models for molecular imaging have been generated that permit the elimination of signal from nonadherent agent.

Nanosized Contrast Agents in Ultrasound Molecular Imaging.

Applying nanosized ultrasound contrast agents (nUCAs) in molecular imaging has received considerable attention. nUCAs have been instrumental in ultrasound molecular imaging to enhance sensitivity, identification, and quantification. nUCAs can achieve high performance in molecular imaging, which was influenced by synthetic formulations and size. This review presents an overview of nUCAs from different synthetic formulations with a discussion on imaging and detection technology. Then we also review the progress of nUCAs in preclinical application and highlight the recent challenges of nUCAs.

Detection and Characterization of Sentinel Lymph Node by Ultrasound Molecular Imaging with B7-H3-Targeted Microbubbles in Orthotopic Breast Cancer Model in Mice.

Accurate identification and assessment of sentinel lymph node (SLN) using noninvasive imaging methods can play a vital role in tumor staging, surgical planning, and prognostic evaluation. In this study, we assessed the efficacy of B7-H3-targeted molecular-ultrasound imaging for the early SLN detection, and characterization in a mouse model of orthotopic breast cancer. We established a mouse breast cancer model with lymph node metastasis by injecting MAD-MB 231 cells which were engineered to express firefly luciferase reporter gene into the fat pad of the right 4th mammary gland in female BALB/c nude mice. The sole lymph node (LN) close to the tumor was regarded as the SLN for imaging investigation, which included metastatic and non-metastatic SLNs. The LN in the right 4th mammary gland from normal mice was used as normal control (normal mice LN). The commercially available preclinical streptavidin-coated, perfluorocarbon-containing lipid-shelled microbubbles (VisualSonics, Toronto, Canada) were used to generate B7-H3-targeted microbubbles (MBB7-H3) and control microbubbles (MBControl). Then, ultrasound molecular imaging (USMI) was performed using a high-resolution transducer (MS250; center frequency, 21MHz; Vevo 2100; VisualSonics, Toronto, Canada) after intravenous injection of microbubbles. The SLN was clearly detected and located under conventional (B-mode) and contrast-enhanced ultrasonography with microbubble injection. The metastatic SLNs showed a markedly higher signal from B7-H3-targeted microbubbles (MBB7-H3) compared to the non-metastatic SLNs and normal LNs. The metastatic SLN was further confirmed by ex vivo bioluminescence imaging and eventually verified by histological analysis. Our findings suggest the potential value of USMI using B7-H3 targeted microbubbles in breast cancer and establish an effective imaging method for the non-invasive detection and characterization of SLN.

Intracolic ultrasound molecular imaging: a novel method for assessing colonic tumor necrosis factor-\u03b1 expression in inflammatory bowel disease

BackgroundWhile anti-tumor necrosis factor alpha (TNF-α) therapy has been proven effective in inflammatory bowel disease (IBD), approximately 40% of patients lose the response. Transmembrane TNF-α (mTNF-α) expression in the intestinal mucosa is correlated with therapeutic efficacy, and quantification of mTNF-α expression is significant for predicting response. However, conventional intravenous application of microbubbles is unable to assess mTNF-α expression in intestinal mucosa. Herein, we proposed intracolic ultrasound molecular imaging with TNF-α-targeted microbubbles (MBTNF-α) to quantitatively detect mTNF-α expression in the intestinal mucosa.MethodsMBTNF-α was synthesized via a biotin–streptavidin bridging method. TNF-α-targeted ultrasound imaging was performed by intracolic application of MBTNF-α to detect mTNF-α expression in surgical specimens from a murine model and patients with IBD. Linear regression analyses were performed to confirm the accuracy of quantitative targeted ultrasound imaging.ResultsOn quantitative TNF-α-targeted ultrasound images, a greater signal intensity was observed in the mouse colons with colitis ([1.96 ± 0.45] × 106 a.u.) compared to that of the controls ([0.56 ± 0.21] × 106 a.u., P < 0.001). Targeted US signal intensities and inflammatory lesions were topographically coupled in mouse colons. Linear regression analyses in specimens of mice and patients demonstrated significant correlations between the targeted ultrasound signal intensity and mTNF-α expression (both P < 0.001). Furthermore, TNF-α-targeted ultrasound imaging qualitatively distinguished the varying inflammatory severity in intestinal specimens from IBD patients.ConclusionIntracolic ultrasound molecular imaging with MBTNF-α enables quantitative assessment of mTNF-α expression. It may be a potential tool for facilitating the implementation of personalized medicine in IBD.

Microbubble Functionalization with Platelet Membrane Enables Targeting and Early Detection of Sepsis-Induced Acute Kidney Injury.

The morbidity and mortality of sepsis-induced acute kidney injury (SAKI) remain high. Early detection using molecular ultrasound imaging may reduce mortality and improve the prognosis. Inspired by the intrinsic relationship between platelets and SAKI, platelet membrane-coated hybrid microbubbles (Pla-MBs) are designed for early recognition of SAKI. Pla-MBs are prepared by ultrasound-assisted recombination of liposomes and platelets, consisting of inherent platelet membrane isolated from platelets. By coating with platelet membranes, Pla-MBs are endowed with various adhesive receptors (such as integrin αIIbβ3), providing a benefit for selective adhesion to damaged endothelium in SAKI. In a rat SAKI model, by combining the advantages of molecular ultrasound imaging and platelet membrane, Pla-MBs display platelet-mimicking properties and achieve the early targeted diagnosis of SAKI prior to the regular laboratory markers of kidney function. Moreover, the expression of platelet-binding proteins (von Willebrand factor and fibrinogen) in the kidneys shows consistent results with molecular ultrasound imaging. Together, microbubble functionalization with platelet membranes is diagnostically beneficial for SAKI and might be a promising modality for endothelial injury diseases in the future.

Ultrasound Molecular Imaging for the Guidance of Ultrasound-Triggered Release of Liposomal Doxorubicin and Its Treatment Monitoring in an Orthotopic Prostatic Tumor Model in Rat

Liposome encapsulation of drugs is an interesting approach in cancer therapy to specifically release the encapsulated drug at the desired treatment site. In addition to thermo-, pH-, light-, enzyme- or redox-responsive liposomes, which have had promising results in (pre-) clinical studies, ultrasound-triggered sonosensitive liposomes represent an exciting alternative to locally trigger the release from these cargos. Localized drug release requires precise tumor visualization to produce a targeted and ultrasound stimulus. We used ultrasound molecular imaging (USMI) with BR55, a vascular endothelial growth factor receptor 2 (VEGFR2)-targeted ultrasound contrast agent, to guide ultrasound-triggered release of sonosensitive liposomes encapsulating doxorubicin (L-DXR) in an orthotopic prostatic rodent tumor model. Forty-eight hours after L-DXR injection, local release of doxorubicin was triggered with a confocal ultrasound device with two focused transducers, 1.1-MHz center frequency, and peak positive and negative pressures of 20.5 and 13 MPa at focus. Tumor size decreased by 20% in 2 wk with L-DXR alone (n = 9) and by 70% after treatment with L-DXR and confocal ultrasound (n = 7) (p < 0.01). The effect of doxorubicin on perfusion/vascularity and VEGFR2 expression was evaluated by USMI and immunohistochemistry of CD31 and VEGFR2 and did not reveal differences in perfusion or VEGFR2 expression in the absence or after the triggered release of liposomes. USMI can provide precise guidance for ultrasound-triggered release of liposomal doxorubicin mediated by a confocal ultrasound device; moreover, the combination of B-mode imaging and USMI can help to follow the response of the tumor to the therapy.

Dynamic Filtering of Adherent and Non-adherent Microbubble Signals Using Singular Value Thresholding and Normalized Singular Spectrum Area Techniques

Ultrasound molecular imaging techniques rely on the separation and identification of three types of signals: static tissue, adherent microbubbles and non-adherent microbubbles. In this study, the image filtering techniques of singular value thresholding (SVT) and normalized singular spectrum area (NSSA) were combined to isolate and identify vascular endothelial growth factor receptor 2-targeted microbubbles in a mouse hindlimb tumor model (n = 24). By use of a Verasonics Vantage 256 imaging system with an L12-5 transducer, a custom-programmed pulse inversion sequence employing synthetic aperture virtual source element imaging was used to collect contrast images of mouse tumors perfused with microbubbles. SVT was used to suppress static tissue signals by 9.6 dB while retaining adherent and non-adherent microbubble signals. NSSA was used to classify microbubble signals as adherent or non-adherent with high accuracy (receiver operating characteristic area under the curve [ROC AUC] = 0.97), matching the classification performance of differential targeted enhancement. The combined SVT + NSSA filtering method also outperformed differential targeted enhancement in differentiating MB signals from all other signals (ROC AUC = 0.89) without necessitating destruction of the contrast agent. The results from this study indicate that SVT and NSSA can be used to automatically segment and classify contrast signals. This filtering method with potential real-time capability could be used in future diagnostic settings to improve workflow and speed the clinical uptake of ultrasound molecular imaging techniques.