Decorrelation Time Mapping as an Analysis Tool for Nanobubble-Based Contrast Enhanced Ultrasound Imaging.

Nanobubbles (NBs; ~100-500 nm diameter) are preclinical ultrasound (US) contrast agents that expand applications of contrast enhanced US (CEUS). Due to their sub-micron size, high particle density, and deformable shell, NBs in pathological states of heightened vascular permeability (e.g. in tumors) extravasate, enabling applications not possible with microbubbles (~1000-10,000 nm diameter). A method that can separate intravascular versus extravascular NB signal is needed as an imaging biomarker for improved tumor detection. We present a demonstration of decorrelation time (DT) mapping for enhanced tumor NB-CEUS imaging. In vitro models validated the sensitivity of DT to agent motion. Prostate cancer mouse models validated in vivo imaging potential and sensitivity to cancerous tissue. Our findings show that DT is inversely related to NB motion, offering enhanced detail of NB dynamics in tumors, and highlighting the heterogeneity of the tumor environment. Average DT was high in tumor regions (~9 s) compared to surrounding normal tissue (~1 s) with higher sensitivity to tumor tissue compared to other mapping techniques. Molecular NB targeting to tumors further extended DT (11 s) over non-targeted NBs (6 s), demonstrating sensitivity to NB adherence. From DT mapping of in vivo NB dynamics we demonstrate the heterogeneity of tumor tissue while quantifying extravascular NB kinetics and delineating intra-tumoral vasculature. This new NB-CEUS-based biomarker can be powerful in molecular US imaging, with improved sensitivity and specificity to diseased tissue and potential for use as an estimator of vascular permeability and the enhanced permeability and retention (EPR) effect in tumors.

Ultrasound-mediated drug-free theranostics for treatment of prostate cancer.

Lipid-shelled nanobubbles (NBs) can be visualized and activated using noninvasive ultrasound (US) stimulation, leading to significant bioeffects. Prior work demonstrates that active targeting of NBs to prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer (PCa) results in enhanced cellular internalization and prolongs NB retention with persistent, cancer-cell specific acoustic activity. In this work, we hypothesized that tumor-accumulated PSMA-NBs combined with low frequency unfocused therapeutic US (TUS) will lead to selective damage and induce a specific therapeutic effect in PSMA-expressing tumors compared to PSMA-negative tumors. We observed that the internalized NBs and cellular compartments were disrupted after the PSMA-NB + TUS (targeted NB therapy or TNT) application, yet treated cells remained intact and viable. In vivo, PSMA-expressing tumors in mice receiving TNT treatment demonstrated a significantly greater extent of apoptosis (78.4 ± 9.3 %, p < 0.01) compared to controls. TNT treatment significantly inhibited the PSMA expressing tumor growth and increased median survival time by 103 %, p < 0.001). A significant reduction in tumor progression compared to untreated control was also seen in an orthotopic rabbit PCa model. Results demonstrate that cavitation of PSMA-NBs internalized via receptor-mediated endocytosis into target PCa cells using unfocused ultrasound results in significant, tumor-specific bioeffects. The effects, while not lethal to PSMA-expressing cancer cells in vitro, result in significant in vivo reduction in tumor progression in two models of PCa. While the mechanism of action of these effects is yet unclear, it is likely related to a locally-induced immune response, opening the door to future investigations in this area.

Quantification of extravasation and binding of PSMA-targeted nanobubbles by modelling the second-wave phenomenon.

PURPOSE: With about ten-fold smaller diameter than MBs, nanobubbles (NBs) were developed as new-generation ultrasound contrast agents (UCA) able to extravasate and target specific receptors expressed on extravascular cancer cells, such as the prostate-specific membrane antigen (PSMA). It has been shown that PSMA-targeted NBs (PSMA-NBs) can bind to specific prostate cancer (PCa) cells and exhibit a prolonged retention effect (PRE), observable by NB-based CEUS (NB-CEUS). However, previous analyses of PRE were mainly limited to the semi-quantitative assessment of the time-intensity curve (TIC) in an entire tumor ROI, possibly losing information on tumor spatial heterogeneity and local characteristics. When analyzing the pixel-level TICs of free NB-based CEUS, we observed a unique second-wave phenomenon: The first pass of the NB wave (bolus) is usually accompanied by a second wave in the time range of 3 to 15 min after the bolus injection. Such a phenomenon was shown to be potentially valuable in supporting the diagnostics of cancerous lesions. PROCEDURES: Seven male athymic nude mice were included and implanted with a tumor expressing PSMA (PSMA+) and tumors not expressing PSMA (PSMA-) on two flanks. Using either free NBs or PSMA-NBs, the characteristics of pixel-level TICs were estimated by a specialized model accounting for the two-wave phenomenon, compared with a conventional model describing only one wave. The estimated parameters by the two models were presented as parametric maps to visualize the PRE of PSMA-NBs in a dual-tumor mouse model. The effectiveness of the two models were also assessed by comparing the estimated parameters in the PSMA+ and PSMA- tumors through Mann-Whitney U test and quartile difference. RESULTS: Two parameters, the peak time and residual factor of the second wave, by the second-wave model were significantly different between PSMA+ and PSMA- tumors when using PSMA-NBs. Compared with the TICs of free NBs, TICs of PSMA-NBs present higher peak intensity and a more delayed second wave, especially in the PSMA+ tumor. CONCLUSIONS: The estimation of parametric maps allows the estimation and visualization of specific binding of PSMA-NBs in PCa. The incorporation of the second-wave phenomenon enrich our understanding of NB kinetics in vivo and can possibly contribute to improved diagnostics of PCa in the future.

Assessing Tumor Microenvironment Characteristics and Stratifying EPR with a Nanobubble Companion Nanoparticle via Contrast-Enhanced Ultrasound Imaging.

The tumor microenvironment is characterized by dysfunctional endothelial cells, resulting in heightened vascular permeability. Many nanoparticle-based drug delivery systems attempt to use this enhanced permeability combined with impaired lymphatic drainage (a concept known as the 'enhanced permeability and retention effect' or EPR effect) as the primary strategy for drug delivery, but this has not proven to be as clinically effective as anticipated. The specific mechanisms behind the inconsistent clinical outcomes of nanotherapeutics have not been clearly articulated, and the field has been hampered by a lack of accessible tools to study EPR-associated phenomena in clinically relevant scenarios. While medical imaging has tremendous potential to contribute to this area, it has not been broadly explored. This work examines, for the first time, the use of multiparametric dynamic contrast-enhanced ultrasound (CEUS) with a novel nanoscale contrast agent to examine tumor microenvironment characteristics noninvasively and in real-time. We demonstrate that CEUS imaging can: (1) evaluate tumor microenvironment features and (2) be used to help predict the distribution of doxorubicin-loaded liposomes in the tumor parenchyma. CEUS using nanobubbles (NBs) was carried out in two tumor types of high (LS174T) and low (U87) vascular permeability, and time-intensity curve (TIC) parameters were evaluated in both models prior to injection of doxorubicin liposomes. Consistently, LS174T tumors showed significantly different TIC parameters, including area under the rising curve (2.7x), time to peak intensity (1.9x) and decorrelation time (DT, 1.9x) compared to U87 tumors. Importantly, the DT parameter successfully predicted tumoral nanoparticle distribution (r = 0.86 ± 0.13). Ultimately, substantial differences in NB-CEUS generated parameters between LS174T and U87 tumors suggest that this method may be useful in determining tumor vascular permeability and could be used as a biomarker for identifying tumor characteristics and predicting sensitivity to nanoparticle-based therapies. These findings could ultimately be applied to predicting treatment efficacy and to evaluating EPR in other diseases with pathologically permeable vasculature.

Ultrasound-mediated drug-free theranostics for treatment of prostate cancer.

Rationale: Lipid-shelled nanobubbles (NBs) can be visualized and activated using noninvasive ultrasound (US) stimulation, leading to significant bioeffects. We have previously shown that active targeting of NBs to prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer (PCa) enhances the cellular internalization and prolongs retention of NBs with persistent acoustic activity (~hrs.). In this work, we hypothesized that tumor-accumulated PSMA-NBs combined with low frequency therapeutic US (TUS) will lead to selective damage and induce a therapeutic effect in PSMA-expressing tumors compared to PSMA-negative tumors. Methods: PSMA-targeted NBs were formulated by following our previously established protocol. Cellular internalization of fluorescent PSMA-NBs was evaluated by confocal imaging using late endosome/lysosome staining pre- and post-TUS application. Two animal models were used to assess the technique. Mice with dual tumors (PSMA expressing and PSMA negative) received PSMA-NB injection via the tail vein followed by TUS 1 hr. post injection (termed, targeted NB therapy or TNT). Twenty-four hours after treatment mice were euthanized and tumor cell apoptosis evaluated via TUNEL staining. Mice with single tumors (either PSMA + or -) were used for survival studies. Tumor size was measured for 80 days after four consecutive TNT treatments (every 3 days). To test the approach in a larger model, immunosuppressed rabbits with orthotopic human PSMA expressing tumors received PSMA-NB injection via the tail vein followed by TUS 30 min after injection. Tumor progression was assessed via US imaging and at the end point apoptosis was measured via TUNEL staining. Results: In vitro TNT studies using confocal microscopy showed that the internalized NBs and cellular compartments were disrupted after the TUS application, yet treated cells remained intact and viable. In vivo, PSMA-expressing tumors in mice receiving TNT treatment demonstrated a significantly greater extent of apoptosis (78.45 ± 9.3%, p < 0.01) compared to the other groups. TNT treatment significantly inhibited the PSMA (+) tumor growth and overall survival significantly improved (median survival time increase by 103%, p < 0.001). A significant reduction in tumor progression compared to untreated control was also seen in the rabbit model in intraprostatic (90%) and in extraprostatic lesions (94%) (p = 0.069 and 0.003, respectively). Conclusion: We demonstrate for the first time the effect of PSMA-targeted nanobubble intracellular cavitation on cancer cell viability and tumor progression in two animal models. Data demonstrate that the targeted nanobubble therapy (TNT) approach relies primarily on mechanical disruption of intracellular vesicles and the resulting bioeffects appear to be more specific to target cancer cells expressing the PSMA receptor. The effect, while not lethal in vitro, resulted in significant tumor apoptosis in vivo in both a mouse and a rabbit model of PCa. While the mechanism of action of these effects is yet unclear, it is likely related to a locally-induced immune response, opening the door to future investigations in this area.

Pharmacokinetic Modeling of the Second-Wave Phenomenon in Nanobubble-Based Contrast-Enhanced Ultrasound.

With a typical 100-500 nm diameter, nanobubbles are a promising new-generation ultrasound contrast agent that paves ways for several applications, such as efficient drug delivery, molecular imaging, and assessment of vascular permeability. Due to their unique physical properties, nanobubbles exhibit distinct in vivo pharmacokinetics. We have shown that the first pass of the nanobubble bolus is usually accompanied by the appearance of a second bolus (wave) within a time range of about 15 minutes. Such phenomenon, to the best of our knowledge, has never been observed with conventional microbubbles and smaller molecular contrast agents used in MRI and CT. In a previous study, we showed the potential of this phenomenon in supporting cancer diagnosis. This study focuses on developing a new compartmental pharmacokinetic model that can be used to interpret the second-wave phenomenon. With this model, we can analyze more in-depth the roles of several physiological factors affecting the characteristics of the second-wave phenomenon.

Pharmacokinetic modeling of PSMA-targeted nanobubbles for quantification of extravasation and binding in mice models of prostate cancer.

PURPOSE: Contrast-enhanced ultrasound (CEUS) by injection of microbubbles (MBs) has shown promise as a cost-effective imaging modality for prostate cancer (PCa) detection. More recently, nanobubbles (NBs) have been proposed as novel ultrasound contrast agents. Unlike MBs, which are intravascular ultrasound contrast agents, the smaller diameter of NBs allows them to cross the vessel wall and target specific receptors on cancer cells such as the prostate-specific membrane antigen (PSMA). It has been demonstrated that PSMA-targeted NBs can bind to the receptors of PCa cells and show a prolonged retention effect in dual-tumor mice models. However, the analysis of the prolonged retention effect has so far been limited to qualitative or semi-quantitative approaches. METHODS: This work introduces two pharmacokinetics models for quantitative analysis of time-intensity curves (TICs) obtained from the CEUS loops. The first model is based on describing the vascular input by the modified local density random walk (mLDRW) model and independently interprets TICs from each tumor lesion. Differently, the second model is based on the reference-tissue model, previously proposed in the context of nuclear imaging, and describes the binding kinetics of an indicator in a target tissue by using a reference tissue where binding does not occur. RESULTS: Our results show that four estimated parameters, ß, ß / λ $\beta /\lambda $ , ß + / ß - ${\beta }_ + /{\beta }_ - $ , for the mLDRW-input model, and γ for the reference-based model, were significantly different (p-value <0.05) between free NBs and PSMA-NBs. These parameters estimated by the two models demonstrate different behaviors between PSMA-targeted and free NBs. CONCLUSIONS: These promising results encourage further quantitative analysis of targeted NBs for improved cancer diagnostics and characterization.

The unique second wave phenomenon in contrast enhanced ultrasound imaging with nanobubbles.

Investigation of nanobubble (NB) pharmacokinetics in contrast-enhanced ultrasound (CEUS) at the pixel level shows a unique phenomenon where the first pass of the contrast agent bolus is accompanied by a second wave. This effect has not been previously observed in CEUS with microbubbles. The objective of this study was to investigate this second-wave phenomenon and its potential clinical applications. Seven mice with a total of fourteen subcutaneously-implanted tumors were included in the experiments. After injecting a bolus of NBs, the NB-CEUS images were acquired to record the time-intensity curves (TICs) at each pixel. These TICs are fitted to a pharmacokinetic model which we designed to describe the observed second-wave phenomenon. The estimated model parameters are presented as parametric maps to visualize the characteristics of tumor lesions. Histological analysis was also conducted in one mouse to compare the molecular features of tumor tissue with the obtained parametric maps. The second-wave phenomenon is evidently shown in a series of pixel-based TICs extracted from either tumor or tissues. The value of two model parameters, the ratio of the peak intensities of the second over the first wave, and the decay rate of the wash-out process present large differences between malignant tumor and normal tissue (0.04 < Jessen-Shannon divergence < 0.08). The occurrence of a second wave is a unique phenomenon that we have observed in NB-CEUS imaging of both mouse tumor and tissue. As the characteristics of the second wave are different between tumor and tissue, this phenomenon has the potential to support the diagnosis of cancerous lesions.

Extrusion: A New Method for Rapid Formulation of High-Yield, Monodisperse Nanobubbles.

Shell-stabilized gas microbubbles (MB) and nanobubbles (NB) are frequently used for biomedical ultrasound imaging and therapeutic applications. While it is widely recognized that monodisperse bubbles can be more effective in these applications, the efficient formulation of uniform bubbles at high concentrations is difficult to achieve. Here, it is demonstrated that a standard mini-extruder setup, commonly used to make vesicles or liposomes, can be used to quickly and efficiently generate monodisperse NBs with high yield. In this highly reproducible technique, the NBs obtained have an average diameter of 0.16 ± 0.05 µm and concentration of 6.2 ± 1.8 × 1010  NBs mL-1 compared to 0.32 ± 0.1 µm and 3.2 ± 0.7 × 1011  mL-1 for NBs made using mechanical agitation. Parameters affecting the extrusion and NB generation process including the temperature, concentration of the lipid solution, and the number of passages through the extruder are also examined. Moreover, it is demonstrated that extruded NBs show a strong acoustic response in vitro and a strong and persistent US signal enhancement under nonlinear contrast enhanced ultrasound imaging in mice. The extrusion process is a new, efficient, and scalable technique that can be used to easily produce high yield smaller monodispersed nanobubbles.

Intracellular vesicle entrapment of nanobubble ultrasound contrast agents targeted to PSMA promotes prolonged enhancement and stability in vivo and in vitro.

Gas-core nanoscale bubbles (or nanobubbles) have gained significant recent attention as promising contrast agents for cancer molecular imaging using medical ultrasound. Previous work has shown that active targeting of nanobubbles to tumor biomarkers such as the prostate-specific membrane antigen (PSMA) significantly prolongs ultrasound signal enhancement, which is a critical feature for successful tumor diagnosis. However, the specific mechanism behind this effect is not well understood, and has not been previously studied in detail. Thus, in the current work, we investigated the process of PMSA- targeted nanobubble transport in tumors across different scales from in vivo whole tumor imaging using high-frequency dynamic contrast-enhanced ultrasound to intracellular confocal imaging and, molecularly using headspace gas chromatography/mass spectrometry. Data demonstrated that, indeed, molecular targeting of nanobubbles to the PSMA biomarker prolongs their tumor uptake and retention across the entire tumor volume, but with variability due to the expected tumor heterogeneity. Importantly, in vitro, the active targeting of NBs results in internalization via receptor-mediated endocytosis into the target cells, and the co-localization with intracellular vesicles (late-stage endosomes/lysosomes) significantly prolongs perfluorocarbon gas retention within the cells. This has not been directly observed previously. These results support the potential for nanobubbles to enable highly specific, background-free diagnostic imaging of the target cells/tissues using ultrasound.

Ultrasound-Based Molecular Imaging of Tumors with PTPmu Biomarker-Targeted Nanobubble Contrast Agents.

Ultrasound imaging is a widely used, readily accessible and safe imaging modality. Molecularly-targeted microbubble- and nanobubble-based contrast agents used in conjunction with ultrasound imaging expand the utility of this modality by specifically targeting and detecting biomarkers associated with different pathologies including cancer. In this study, nanobubbles directed to a cancer biomarker derived from the Receptor Protein Tyrosine Phosphatase mu, PTPmu, were evaluated alongside non-targeted nanobubbles using contrast enhanced ultrasound both in vitro and in vivo in mice. In vitro resonant mass and clinical ultrasound measurements showed gas-core, lipid-shelled nanobubbles conjugated to either a PTPmu-directed peptide or a Scrambled control peptide were equivalent. Mice with heterotopic human tumors expressing the PTPmu-biomarker were injected with PTPmu-targeted or control nanobubbles and dynamic contrast-enhanced ultrasound was performed. Tumor enhancement was more rapid and greater with PTPmu-targeted nanobubbles compared to the non-targeted control nanobubbles. Peak tumor enhancement by the PTPmu-targeted nanobubbles occurred within five minutes of contrast injection and was more than 35% higher than the Scrambled nanobubble signal for the subsequent two minutes. At later time points, the signal in tumors remained higher with PTPmu-targeted nanobubbles demonstrating that PTPmu-targeted nanobubbles recognize tumors using molecular ultrasound imaging and may be useful for diagnostic and therapeutic purposes.

Molecular imaging of orthotopic prostate cancer with nanobubble ultrasound contrast agents targeted to PSMA.

Ultrasound imaging is routinely used to guide prostate biopsies, yet delineation of tumors within the prostate gland is extremely challenging, even with microbubble (MB) contrast. A more effective ultrasound protocol is needed that can effectively localize malignancies for targeted biopsy or aid in patient selection and treatment planning for organ-sparing focal therapy. This study focused on evaluating the application of a novel nanobubble ultrasound contrast agent targeted to the prostate specific membrane antigen (PSMA-targeted NBs) in ultrasound imaging of prostate cancer (PCa) in vivo using a clinically relevant orthotopic tumor model in nude mice. Our results demonstrated that PSMA-targeted NBs had increased extravasation and retention in PSMA-expressing orthotopic mouse tumors. These processes are reflected in significantly different time intensity curve (TIC) and several kinetic parameters for targeted versus non-targeted NBs or LUMASON MBs. These, may in turn, lead to improved image-based detection and diagnosis of PCa in the future.

Real time ultrasound molecular imaging of prostate cancer with PSMA-targeted nanobubbles.

Contrast-enhanced ultrasound with microbubbles has shown promise in detection of prostate cancer (PCa), but sensitivity and specificity remain challenging. Targeted nanoscale-contrast agents with improved capability to accumulate in tumors may result in prolonged signal enhancement and improved detection of PCa with ultrasound. Here we report nanobubbles (NB) that specifically targets prostate specific membrane antigen (PSMA) overexpressed in PCa. The PSMA-targeted-NB (PSMA-NB) were utilized to simultaneously image dual-flank PCa (PSMA-positive PC3pip and PSMA-negative PC3flu) to examine whether the biomarker can be successfully detected and imaged in a mouse model. Results demonstrate that active targeting rapidly and selectively enhances tumor accumulation and tumor retention. Importantly, these processes could be visualized and quantified, in real-time, with clinical ultrasound. Such demonstration of the immense yet underutilized potential of ultrasound in the molecular imaging area can open the door to future opportunities for improving sensitivity and specificity of cancer detection using parametric NB-enhanced ultrasound imaging.

Contrast enhanced ultrasound imaging by nature-inspired ultrastable echogenic nanobubbles.

Advancement of ultrasound molecular imaging applications requires not only a reduction in size of the ultrasound contrast agents (UCAs) but also a significant improvement in the in vivo stability of the shell-stabilized gas bubble. The transition from first generation to second generation UCAs was marked by an advancement in stability as air was replaced by a hydrophobic gas, such as perfluoropropane and sulfur hexafluoride. Further improvement can be realized by focusing on how well the UCAs shell can retain the encapsulated gas under extreme mechanical deformations. Here we report the next generation of UCAs for which we engineered the shell structure to impart much better stability under repeated prolonged oscillation due to ultrasound, and large changes in shear and turbulence as it circulates within the body. By adapting an architecture with two layers of contrasting elastic properties similar to bacterial cell envelopes, our ultrastable nanobubbles (NBs) withstand continuous in vitro exposure to ultrasound with minimal signal decay and have a significant delay on the onset of in vivo signal decay in kidney, liver, and tumor. Development of ultrastable NBs can potentially expand the role of ultrasound in molecular imaging, theranostics, and drug delivery.

Time-intensity-curve Analysis and Tumor Extravasation of Nanobubble Ultrasound Contrast Agents.

Our group recently presented a simple strategy using the non-ionic surfactant, Pluronic, as a size control excipient to produce nanobubbles in the 100-nm range, which exhibited stability and echogenicity on par with clinically available microbubbles. The objective of the present study was to evaluate biodistribution and extravasation of the Pluronic-stabilized lipid nanobubbles compared with microbubbles in 2 experimental tumor models in mice. Standard lipid-stabilized perfluoropropane bubbles (Pluronic L10) and lipid-stabilized perfluoropropane nanobubbles were intravenously injected into mice bearing either an orthotopic mouse breast cancer (BC4 T1) or subcutaneous mouse ovarian cancer (OVCAR-3) through the tail vein to perform perfusion dynamic studies. No significant differences between the nanobubble and microbubble groups were observed in the peak enhancement of the 3 tested regions (tumor, liver and kidney). However, the decay rates of nanobubble in the tumor and kidney of BC4 T1-bearing mice, as well as in mice with OVRCAR-3 tumors were significantly slower than those of the microbubble. To quantify extravasation, fluorescently labeled bubbles were intravenously injected into mice bearing the same tumors. Histologic analysis showed that nanobubbles were retained in tumor tissue to a greater extent compared with microbubbles in both tumor models at the 3-h time point. Our results demonstrate unique nanobubble behavior compared with microbubbles and support augmented application of these agents in ultrasound molecular imaging and drug delivery beyond the tumor vasculature.

Effect of Bubble Concentration on the in Vitro and in Vivo Performance of Highly Stable Lipid Shell-Stabilized Micro- and Nanoscale Ultrasound Contrast Agents.

Ultrasound (US) is a widely used diagnostic imaging tool because it is inexpensive, safe, portable, and broadly accessible. Ultrasound contrast agents (UCAs) are employed to enhance backscatter echo and improve imaging contrast. The most frequently utilized UCAs are echogenic bubbles made with a phospholipid or protein-stabilized hydrophobic gas core. While clinically utilized, applications of UCAs are often limited by rapid signal decay (<5 min) in vivo under typical ultrasound imaging protocols. Here, we report on a formulation of lipid shell-stabilized perfluoropropane (C3F8) microbubbles and nanobubbles with a significantly prolonged in vivo stability. Microbubbles (875 ± 280 nm) of the target size were prepared by utilizing a multiple-step centrifugation cycle, while nanobubbles (299 ± 189 nm) were isolated from the activated vial using a single centrifugation step. To provide in-depth acoustic characterization of the new construct we evaluated the effect of size and concentration on their in vitro and in vivo performance. In vitro and in vivo characterization were carried out for a range of bubble concentrations normalized by total gas volume quantified via headspace gas chromatography/mass spectrometry (GC/MS). In vitro characterization revealed that nanobubbles at different concentrations are more consistently stable over time with the highest and lowest dilutions (50-fold decrease) only differing in US signal after 8 min exposure by 10.34%, while for microbubbles the difference was 86.46%. As expected, due to the difference in hydrodynamic diameter and scattering cross section difference, nanobubbles showed lower overall initial signal intensity. In vivo experiments showed that both microbubbles and nanobubbles with similar initial peak signal intensity are comparably stable over time with 66.8% and 60.6% remaining signal after 30 min, respectively. This study demonstrates that bubble concentration has significant effects on the persistence of both microbubbles and nanobubbles in vitro and in vivo, but the effects are more pronounced in larger bubbles. These effects should be taken into account when selecting the appropriate bubble parameters for future imaging applications.

Sink or float? Characterization of shell-stabilized bulk nanobubbles using a resonant mass measurement technique.

Nano-sized shell-stabilized gas bubbles have applications in various fields ranging from environmental science to biomedical engineering. A resonant mass measurement (RMM) technique is demonstrated here as a new and only method capable of simultaneously measuring the size and concentration of buoyant and non-buoyant particles in a nanobubble sample used as a next-generation ultrasound contrast agent.

Acoustic Actuation of Integrin-Bound Microbubbles for Mechanical Phenotyping during Differentiation and Morphogenesis of Human Embryonic Stem Cells.

Early human embryogenesis is a dynamic developmental process, involving continuous and concomitant changes in gene expression, structural reorganization, and cellular mechanics. However, the lack of investigation methods has limited the understanding of how cellular mechanical properties change during early human embryogenesis. In this study, ultrasound actuation of functionalized microbubbles targeted to integrin (acoustic tweezing cytometry, ATC) is employed for in situ measurement of cell stiffness during human embryonic stem cell (hESC) differentiation and morphogenesis. Cell stiffness, which is regulated by cytoskeleton structure, remains unchanged in undifferentiated hESCs, but significantly increases during neural differentiation. Further, using the recently established in vitro 3D embryogenesis models, ATC measurements reveal that cells continue to stiffen while maintaining pluripotency during epiblast cyst formation. In contrast, during amniotic cyst formation, cells first become stiffer during luminal cavity formation, but softens significantly when cells differentiate to form amniotic cysts. These results suggest that cell stiffness changes not only due to 3D spatial organization, but also with cell fate change. ATC therefore provides a versatile platform for in situ measurement of cellular mechanical property, and cell stiffness may be used as a mechanical biomarker for cell lineage diversification and cell fate specification during embryogenesis.

Ultrasound Contrast Agents and Delivery Systems in Cancer Detection and Therapy.

Ultrasound is the second most utilized imaging modality in the world because it is widely accessible, robust, and safe. Aside from its extensive use in diagnostic imaging, ultrasound has also been frequently utilized in therapeutic applications. Particularly, when combined with appropriate delivery systems, ultrasound provides a flexible platform for simultaneous real-time imaging and triggered release, enabling precise, on-demand drug delivery to target sites. This chapter will discuss the basics of ultrasound including its mechanism of action and how it can be used to trigger the release of encapsulated drug either through thermal or cavitation effects. Fundamentals of ultrasound contrast agents, how they enhance ultrasound signals, and how they can be modified to function as carriers for triggered and targeted release of drugs will also be discussed.

Improving performance of nanoscale ultrasound contrast agents using N,N-diethylacrylamide stabilization.

The design of nanoscale yet highly echogenic agents for imaging outside of the vasculature and for ultrasound-mediated drug delivery remains a formidable challenge. We have previously reported on formulation of echogenic perfluoropropane gas nanobubbles stabilized by a lipid-pluronic surfactant shell. In the current work we describe the development of a new generation of these nanoparticles which consist of perfluoropropane gas stabilized by a surfactant and lipid membrane and a crosslinked network of N,N-diethylacrylamide. The resulting crosslinked nanobubbles (CL-PEG-NB) were 95.2±25.2nm in diameter and showed significant improvement in stability and retention of echogenic signal over 24h. In vivo analysis via ultrasound and fluorescence mediated tomography showed greater tumor extravasation and accumulation with CL-PEG-NB compared to microbubbles. Together these results demonstrate the capabilities and advantages of a new, more stable, nanometer-scale ultrasound contrast agent that can be utilized in future work for diagnostic scans and molecular imaging.