The Effect of Additives on the Behavior of Phase Sensitive In Situ Forming Implants

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113738/1/jps24558.pd

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

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 actuation of functionalized microbubbles targeted to integrin (acoustic tweezing cytometry) is employed for in situ measurement of cell stiffness during human embryonic stem cell neural differentiation and morphogenesis in 3D embryogenesis model. The results suggest that cell stiffness changes not only due to 3D spatial organization, but also with cell fate change.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146940/1/smll201803137.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146940/2/smll201803137_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146940/3/smll201803137-sup-0001-S1.pd

Porphyrin-Loaded Pluronic Nanobubbles: A New US-Activated Agent for Future Theranostic Applications

Sonodynamic therapy (SDT) has become a promising noninvasive approach for cancer therapy. The treatment exploits the ability of particular molecules (i.e., porphyrins) to be excited by ultrasound and produce reactive oxygen species (ROS) during their decay process. These reactive species, in turn, result in cell death. To capitalize on the real-time visualization and on-demand delivery of ultrasound contrast agents, this study aims to combine porphyrins with nanobubbles (NBs) to obtain an ultrasound-activated theranostic agent that exploits the SDT activity <i>in vitro</i>. Two porphyrin classes, exposing different hydrophobic side chains, were synthesized. NB size and encapsulation efficiency were markedly dependent on the porphyrin structure. The combination of these porphyrin and NBs resulted in a significant reduction in cell viability upon sonication in pilot studies performed on the LS 174T colorectal cancer cell line

Pharmacokinetic analysis of targeted nanonubbles for quantitative assessment of PSMA expression in prostate cancer

Despite showing promise, the sensitivity of contrast enhanced ultrasound (CEUS) for prostate cancer diagnosis is still limited. The introduction of novel microbubbles (MBs) targeted to the vascualar endhotelial growth receptor factor 2 has opened new possibilites for molecular imaging of prostate cancer. However, the detection rate in a phase-0 clinical trial was still limited to 65%. More effective contrast agents are needed to improve diagnostic accuracy. Recently, ultrasound nanobubbles (NBs) are emerging as promising agents for improved cancer diagnostics and therapy. Thanks to their reduced diameter, which is about 10 times smaller than MBs, they can cross the vascular endhotelium, providing greater possibilities for targeted imaging and therapy, including targets no longer limited to the vessel wall. In this context, a long-circulating NB targeted to the prostate-specific membrane antigent (PSMA) was recently developed, showing promise for selective accumulation in tumors expressing PSMA. In this work, we propose pharmacokinetic modeling of the kinetics of PSMA-targeted NBs by the simplified reference tissue model. While this model has been originially developed for receptor kinetic studies in nuclear medicine, it is here adapted for CEUS, enabling quantitative assessment of PSMA expression by estimation of the binding potential BP. The model is validated by comparing the estimated binding parameter obtained for three different US contrast agents (conventional MBs, non-targeted NBs, and PSMA-targeted NBs) in a dual tumor mouse model, carrying a PSMA-positive tumor in one flank, and a PSMA-negative tumor in the other flank

Quantification of PSMA expression in prostate cancer by pharmacokinetic modeling of targeted ultrasound nanobubbles

The value of contrast-enhanced ultrasound (CEUS) for prostate cancer diagnostics is still debated. Novel targeted ultrasound contrast agents enable visualization of molecular and cellular processes in vivo and non-invasively. Microbubbles targeted to the vascular endothelial growth factor receptor 2 have been successfully tested in humans, but detection rate for prostate cancer was limited to 65%. While microbubbles can only target molecules in the blood vessels, novel nanobubbles (NBs) can extravasate, thus enabling reaching targets beyond the vessel wall. Recently, NBs targeted to the prostate specific membrane antigen (PSMA), which is overexpressed by prostate cancer cells, have shown selective accumulation in prostate-tumor mouse models. However, methods for quantification of NB binding are still lacking. In this work, we propose a pharmacokinetic modeling approach to estimate the binding potential of PSMA-targeted NBs, and we test the proposed method in 7 dual-tumor mouse models of prostate cancer

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, β, (Formula presented.), (Formula presented.), 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