Multispecies comparative prostate anatomy by imaging: Implications for experimental models of prostatic disease.

BACKGROUND: There is an increasing interest in using preclinical models for development and assessment of medical devices and imaging techniques for prostatic disease care. Still, a comprehensive assessment of the prostate's radiological anatomy in primary preclinical models such as dogs, rabbits, and mice utilizing human anatomy as a reference point remains necessary with no optimal model for each purpose being clearly defined in the literature. Therefore, this study compares the anatomical characteristics of different animal models to the human prostatic gland from the imaging perspective. METHODS: We imaged five Beagle laboratory dogs, five New Zealand White rabbits, and five mice, all sexually mature males, under Institutional Animal Care and Use Committee (IACUC) approval. Ultrasonography (US) was performed using the Vevo® F2 for mice (57 MHz probe). Rabbits and dogs were imaged using the Siemens® Acuson S3000 (17 MHz probe) and endocavitary (8 MHz) probes, respectively. Magnetic resonance imaging (MRI) was also conducted with a 7T scanner in mice and 3T scanner in rabbits and dogs. RESULTS: Canine transrectal US emerged as the optimal method for US imaging, depicting a morphologically similar gland to humans but lacking echoic zonal differentiation. MRI findings in canines indicated a homogeneously structured gland similar to the human peripheral zone on T2-weighted images (T2W) and apparent diffusion coefficient (ADC). In rabbits, US imaging faced challenges due to the pubic symphysis, whereas MRI effectively visualized all structures with the prostate presenting a similar aspect to the human peripheral gland on T2W and ADC maps. Murine prostate assessment revealed poor visualization of the prostate glands in ultrasound due to its small size, while 7T MRI delineated the distinct prostates and its lobes, with the lateral and dorsal prostate resembling the peripheral zone and the anterior prostate the central zone of the human gland. CONCLUSION: Dogs stand out as superior models for advanced preclinical studies in prostatic disease research. However, mice present as a good model for early stage studies and rabbits are a cost-effective alternative and serve as valuable tools in specific research domains when canine research is not feasible.

Development of a novel castration-resistant orthotopic prostate cancer model in New Zealand White rabbit.

BACKGROUND: Prostate cancer (PCa) models in mice and rats are limited by their size and lack of a clearly delineated or easily accessible prostate gland. The canine PCa model is currently the only large animal model which can be used to test new preclinical interventions but is costly and availability is sparse. As an alternative, we developed an orthotopic human prostate tumor model in an immunosuppressed New Zealand White rabbit. Rabbits are phylogenetically closer to humans, their prostate gland is anatomically similar, and its size allows for clinically-relevant testing of interventions. METHODS: Rabbits were immunosuppressed via injection of cyclosporine. Human PC3pipGFP PCa cells were injected into the prostate via either (a) laparotomy or (b) transabdominal ultrasound (US) guided injection. Tumor growth was monitored using US and magnetic resonance imaging (MRI). Contrast-enhanced ultrasound (CEUS) imaging using nanobubbles and Lumason microbubbles was also performed to examine imaging features and determine the optimal contrast dose required for enhanced visualization of the tumor. Ex vivo fluorescence imaging, histopathology, and immunohistochemistry analyses of the collected tissues were performed to validate tumor morphology and prostate-specific membrane antigen (PSMA) expression. RESULTS: Immunosuppression and tumor growth were, in general, well-tolerated by the rabbits. Fourteen out of 20 rabbits, with an average age of 8 months, successfully grew detectable tumors from Day 14 onwards after cell injection. The tumor growth rate was 39 ± 25 mm2 per week. CEUS and MRI of tumors appear hypoechoic and T2 hypointense, respectively, relative to normal prostate tissue. Minimally invasive US-guided tumor cell injection proved to be a better method compared to laparotomy due to the shorter recovery time required for the rabbits following injection. Among the rabbits that grew tumors, seven had tumors both inside and outside the prostate, three had tumors only inside the prostate, and four had tumors exclusively outside of the prostate. All tumors expressed the PSMA receptor. CONCLUSIONS: We have established, for the first time, an orthotopic PCa rabbit model via percutaneous US-guided tumor cell inoculation. This animal model is an attractive, clinically relevant intermediate step to assess preclinical diagnostic and therapeutic compounds.

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.

Iridium(III) Complex-Loaded Perfluoropropane Nanobubbles for Enhanced Sonodynamic Therapy.

Sonodynamic therapy (SDT) is a novel promising approach for the minimally invasive treatment of cancer derived from photodynamic therapy (PDT). In this study, we have explored an effective sonosensitizer for SDT by loading the iridium(III) complex [Ir(ppy)2(en)] OOCCH3, where ppy = 2-phenylpyridine and en = ethylenediamine], from now on referred to as Ir, with high photosensitizing ability, into echogenic nanobubbles (Ir-NBs). Akin to photosensitizers, sonosensitizers are acoustically activated by deep-tissue-penetrating low-frequency ultrasound (US) resulting in a localized therapeutic effect attributed to an excessive generation of reactive oxygen species (ROS). The Ir-NB formulation was optimized, and the in vitro characterizations were carried out, including physical properties, acoustic performance, intracellular ROS generation, and cytotoxicity against two human cancer cell lines. Ir-NBs had an average size of 303.3 ± 91.7 nm with a bubble concentration of 9.28 × 1010 particles/mL immediately following production. We found that the initial Ir feeding concentration had a negligible effect on the NB size, but affected the bubble concentration as well as the acoustic performance of the NBs. Through a combination of sonication and Ir-NBs treatment, an increase of 68.8% and 69.6% cytotoxicity in human ovarian cancer cells (OVCAR-3) and human breast cancer cells (MCF-7), respectively, was observed compared to the application of Ir-NBs alone. Furthermore, Ir-NBs exposed to the US also induced the highest levels of intracellular ROS generation compared to free Ir and free Ir with empty NBs. The combination of these results suggests that the differences in treatment efficacy is a direct result of acoustic cavitation. These results provide evidence that US activated Ir-loaded NBs have the potential to become an effective sonosensitizer for SDT.

Microfluidic Generation of Monodisperse Nanobubbles by Selective Gas Dissolution.

Nanotechnology currently enables the fabrication of uniform solid nanoparticles and liquid nano-emulsions, but not uniform gaseous nanobubbles (NBs). In this article, for the first time, a method based on microfluidics that directly produces monodisperse NBs is reported. Specifically, a two-component gas mixture of water-soluble nitrogen and water-insoluble octafluoropropane as the gas phase are used in a microfluidic bubble generator. First, monodisperse microbubbles (MBs) with a classical microfluidic flow-focusing junction is generated, then the MBs shrink down to ≈100 nm diameter, due to the dissolution of the water-soluble components in the gas mixture. The degree of shrinkage is controlled by tuning the ratio of water-soluble to water-insoluble gas components. This technique maintains the monodispersity of the NBs, and enables precise control of the final NB size. It is found that the monodisperse NBs show better homogeneity than polydisperse NBs in in vitro ultrasound imaging experiments. Proof-of-concept in vivo kidney imaging is performed in live mice, demonstrating enhanced contrast using the monodisperse NBs. The NB monodispersity and imaging results make microfluidically generated NBs promising candidates as ultrasound contrast and molecular imaging agents.

Bursting Microbubbles: How Nanobubble Contrast Agents Can Enable the Future of Medical Ultrasound Molecular Imaging and Image-Guided Therapy.

The field of medical ultrasound has undergone a significant evolution since the development of microbubbles as contrast agents. However, due to their size, microbubbles remain in the vasculature, and therefore have limited clinical applications. Building a better - and smaller - bubble can expand the applications of contrast-enhanced ultrasound by allowing bubbles to extravasate from blood vessels - creating new opportunities. In this review, we summarize recent research on the formulation and use of NBs as imaging agents and as therapeutic vehicles. We discuss the ongoing debates in the field and reluctance to accepting NBs as an acoustically active construct and a potentially impactful clinical tool that can help shape the future of medical ultrasound. We hope that the overview of key experimental and theoretical findings in the NB field presented in this paper provides a fundamental framework that will help clarify NB-ultrasound interactions and inspire engagement in the field.

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.

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.

Inhibition of the histone demethylase, KDM5B, directly induces re-expression of tumor suppressor protein HEXIM1 in cancer cells.

BACKGROUND: The tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA. METHODS: We identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin-NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays. RESULTS: We have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis. CONCLUSION: HMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.

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.

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.

Polymer Nanosheet Containing Star-Like Copolymers: A Novel Scalable Controlled Release System.

Poly(ε-caprolactone) (PCL)-based nanomaterials, such as nanoparticles and liposomes, have exhibited great potential as controlled release systems, but the difficulties in large-scale fabrication limit their practical applications. Among the various methods being developed to fabricate polymer nanosheets (PNSs) for different applications, such as Langmuir-Blodgett technique and layer-by-layer assembly, are very effort consuming, and only a few PNSs can be obtained. In this paper, poly(ε-caprolactone)-based PNSs with adjustable thickness are obtained in large quantity by simple water exposure of multilayer polymer films, which are fabricated via a layer multiplying coextrusion method. The PNS is also demonstrated as a novel controlled guest release system, in which release kinetics are adjustable by the nanosheet thickness, pH values of the media, and the presence of protecting layers. Theoretical simulations, including Korsmeyer-Peppas model and Finite-element analysis, are also employed to discern the observed guest-release mechanisms.

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 in vitro. 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.

Ultrasound molecular imaging of ovarian cancer with CA-125 targeted nanobubble contrast agents.

Ultrasound is frequently utilized in diagnosis of gynecologic malignancies such as ovarian cancer. Because epithelial ovarian cancer (EOC) is often characterized by overexpression of cancer antigen 125 (CA-125), ultrasound contrast agents able to target this molecular signature could be a promising complementary strategy. In this work, we demonstrate application of CA-125-targeted echogenic lipid and surfactant-stabilized nanobubbles imaged with standard clinical contrast harmonic ultrasound for imaging of CA-125 positive OVCAR-3 tumors in mice. Surface functionalization of the nanobubbles with a CA-125 antibody achieved rapid significantly (P < 0.05) enhanced tumor accumulation, higher peak ultrasound signal intensity and slower wash out rates in OVCAR-3 tumors compared to CA-125 negative SKOV-3 tumors. Targeted nanobubbles also exhibited increased tumor retention and prolonged echogenicity compared to untargeted nanobubbles. Data suggest that ultrasound molecular imaging using CA-125 antibody-conjugated nanobubbles may contribute to improved diagnosis of EOC.

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.

Real-time monitoring of radiofrequency ablation and postablation assessment: accuracy of contrast-enhanced US in experimental rat liver model.

PURPOSE: To examine the accuracy of the unenhanced zone at contrast material-enhanced ultrasonography (US) in predicting coagulative necrosis during and 21 days after radiofrequency (RF) ablation by using radiologic-pathologic comparison. MATERIALS AND METHODS: Animal studies were approved by the Institutional Animal Care and Use Committee. The livers of 28 rats underwent US-guided RF ablation. In four animals, contrast-enhanced US was performed during ablation and 2 hours and 2, 7, 14, and 21 days after ablation. The unenhanced zone area on US images was measured. DiI-labeled microbubbles were administered during ablation at 2, 4, and 6 minutes or at 2 hours and 2, 7, 14, and 21 days after ablation in the remaining 24 animals (n = 3 at each time point). One minute later, the animal was euthanized, and the ablated liver was harvested. Tissue samples were imaged to quantify total fluorescence, and NADH staining was performed on the same slice. Hematoxylin-eosin staining was also performed. The findings on fluorescence images, NADH-stained images, and hematoxylin-eosin-stained images were compared. The areas of DiI bubble-negative zones, NADH-negative zones, and lightly NADH-staining zones were measured. Data were analyzed by using one-way analysis of variance. RESULTS: The area of the unenhanced zone on contrast-enhanced US images increased during RF ablation and reached a maximum within 2 days after ablation. At histopathologic examination, a transition zone manifested adjacent to the coagulation zone until 2 days after ablation. The DiI-bubble negative zone on fluorescence images and the damaged zone (transition zone plus coagulation zone) on NADH-stained images increased rapidly within 2 hours after ablation, then slowly reached the maximum on day 2. The ratios of the mean areas of these two zones at hour 2 to those at day 2 were 94.6% and 95.6%, respectively. High uniformity between the damaged zone on NADH-stained images and the DiI bubble-negative zone on fluorescence images was noted at all time points. CONCLUSION: The temporary transition zone in NADH staining is partially damaged and should transition to nonviability 2 days after ablation. These results demonstrate that contrast-enhanced US can help delineate the maximum area of cell damage (to within 5% of the maximum) as early as 2 hours after ablation. Contrast-enhanced US may be a simple and accurate tool for monitoring the effects of RF ablation and quantifying the size of thermal damage after treatment.

Nanobubble ultrasound contrast agents for enhanced delivery of thermal sensitizer to tumors undergoing radiofrequency ablation.

PURPOSE: Pluronic has been shown to sensitize various tumor cell lines to chemotherapy and hyperthermia by altering the membrane fluidity, depleting ATP, and modulating the heat shock protein 70 expression. In our prior work, Pluronic was also used to formulate nanosized ultrasound contrast agents. In the current study we evaluate the use of these contrast agents as vehicles for image-guided delivery of Pluronic to improve outcomes of tumor radiofrequency (RF) ablation. METHODS: Lipid-shelled Pluronic nanobubbles were prepared and examined for size distribution, zeta potential, stability, biodistribution, accumulation of nanobubbles in the tumor, and treatment efficacy. LS174-T xenograft tumor-bearing mice were used to evaluate tumor growth suppression and measure treatment efficacy after RF ablation. RESULTS: The average diameter of Pluronic bubbles was 230 nm, and initial bubble echogenicity was 16 dB. In vitro, cells exposed to Pluronic nanobubbles exhibited low cytotoxicity in the absence of ultrasound, even if heat (43 ºC) was applied. When the cells were exposed to Pluronic nanobubbles, heat, and ultrasound; viability was significantly reduced. In vivo, tumors treated with ultrasound-modulated nanobubbles prior to RF ablation showed a significant reduction in growth compared to the RF alone (P<0.05). CONCLUSION: Lipid and Pluronic-shelled, echogenic nanobubbles combined with ultrasound modulation can serve as an effective theranostic method for sensitization of tumors to RF ablation.

Using imaging modalities to predict nanoparticle distribution and treatment efficacy in solid tumors: The growing role of ultrasound.

Nanomedicine in oncology has not had the success in clinical impact that was anticipated in the early stages of the field's development. Ideally, nanomedicines selectively accumulate in tumor tissue and reduce systemic side effects compared to traditional chemotherapeutics. However, this has been more successful in preclinical animal models than in humans. The causes of this failure to translate may be related to the intra- and inter-patient heterogeneity of the tumor microenvironment. Predicting whether a patient will respond positively to treatment prior to its initiation, through evaluation of characteristics like nanoparticle extravasation and retention potential in the tumor, may be a way to improve nanomedicine success rate. While there are many potential strategies to accomplish this, prediction and patient stratification via noninvasive medical imaging may be the most efficient and specific strategy. There have been some preclinical and clinical advances in this area using MRI, CT, PET, and other modalities. An alternative approach that has not been studied as extensively is biomedical ultrasound, including techniques such as multiparametric contrast-enhanced ultrasound (mpCEUS), doppler, elastography, and super-resolution processing. Ultrasound is safe, inexpensive, noninvasive, and capable of imaging the entire tumor with high temporal and spatial resolution. In this work, we summarize the in vivo imaging tools that have been used to predict nanoparticle distribution and treatment efficacy in oncology. We emphasize ultrasound imaging and the recent developments in the field concerning CEUS. The successful implementation of an imaging strategy for prediction of nanoparticle accumulation in tumors could lead to increased clinical translation of nanomedicines, and subsequently, improved patient outcomes. This article is categorized under: Diagnostic Tools In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery Emerging Technologies.

Synchronous Intravital Imaging and Cavitation Monitoring of Antivascular Focused Ultrasound in Tumor Microvasculature Using Monodisperse Low Boiling Point Nanodroplets.

Focused ultrasound-stimulated microbubbles can induce blood flow shutdown and ischemic necrosis at higher pressures in an approach termed antivascular ultrasound. Combined with conventional therapies of chemotherapy, immunotherapy, and radiation therapy, this approach has demonstrated tumor growth inhibition and profound synergistic antitumor effects. However, the lower cavitation threshold of microbubbles can potentially yield off-target damage that the polydispersity of clinical agent may further exacerbate. Here we investigate the use of a monodisperse nanodroplet formulation for achieving antivascular effects in tumors. We first develop stable low boiling point monodisperse lipid nanodroplets and examine them as an alternative agent to mediate antivascular ultrasound. With synchronous intravital imaging and ultrasound monitoring of focused ultrasound-stimulated nanodroplets in tumor microvasculature, we show that nanodroplets can trigger blood flow shutdown and do so with a sharper pressure threshold and with fewer additional events than conventionally used microbubbles. We further leverage the smaller size and prolonged pharmacokinetic profile of nanodroplets to allow for potential passive accumulation in tumor tissue prior to antivascular ultrasound, which may be a means by which to promote selective tumor targeting. We find that vascular shutdown is accompanied by inertial cavitation and complex-order sub- and ultraharmonic acoustic signatures, presenting an opportunity for effective feedback control of antivascular ultrasound.