Controlled Tempering of Lipid Concentration and Microbubble Shrinkage as a Possible Mechanism for Fine-Tuning Microbubble Size and Shell Properties.

The acoustic response of microbubbles (MBs) depends on their resonance frequency, which is dependent on the MB size and shell properties. Monodisperse MBs with tunable shell properties are thus desirable for optimizing and controlling the MB behavior in acoustics applications. By utilizing a novel microfluidic method that uses lipid concentration to control MB shrinkage, we generated monodisperse MBs of four different initial diameters at three lipid concentrations (5.6, 10.0, and 16.0 mg/mL) in the aqueous phase. Following shrinkage, we measured the MB resonance frequency and determined its shell stiffness and viscosity. The study demonstrates that we can generate monodisperse MBs of specific sizes and tunable shell properties by controlling the MB initial diameter and aqueous phase lipid concentration. Our results indicate that the resonance frequency increases by 180-210% with increasing lipid concentration (from 5.6 to 16.0 mg/mL), while the bubble diameter is kept constant. Additionally, we find that the resonance frequency decreases by 260-300% with an increasing MB final diameter (from 5 to 12 µm), while the lipid concentration is held constant. For example, our results depict that the resonance frequency increases by ∼195% with increasing lipid concentration from 5.6 to 16.0 mg/mL, for ∼11 µm final diameter MBs. Additionally, we find that the resonance frequency decreases by ∼275% with increasing MB final diameter from 5 to 12 µm when we use a lipid concentration of 5.6 mg/mL. We also determine that MB shell viscosity and stiffness increase with increasing lipid concentration and MB final diameter, and the level of change depends on the degree of shrinkage experienced by the MB. Specifically, we find that by increasing the concentration of lipids from 5.6 to 16.0 mg/mL, the shell stiffness and viscosity of ∼11 µm final diameter MBs increase by ∼400 and ∼200%, respectively. This study demonstrates the feasibility of fine-tuning the MB acoustic response to ultrasound by tailoring the MB initial diameter and lipid concentration.

Microfluidic nanobubbles: observations of a sudden contraction of microbubbles into nanobubbles.

Microfluidic devices are often utilized to generate uniform-size microbubbles. In most microfluidic bubble generation experiments, once the bubbles are formed the gas inside the bubbles begin to dissolve into the surrounding aqueous environment. The bubbles shrink until they attain an equilibrium size dictated by the concentration and type of amphiphilic molecules stabilizing the gas-liquid interface. Here, we exploit this shrinkage mechanism, and control the solution lipid concentration and microfluidic geometry, to make monodisperse bulk nanobubbles. Interestingly, we make the surprising observation of a critical microbubble diameter above and below which the scale of bubble shrinkage dramatically changes. Namely, microbubbles generated with an initial diameter larger than the critical diameter shrinks to a stable diameter that is consistent with previous literature. However, microbubbles that are initially smaller than the critical diameter experience a sudden contraction into nanobubbles whose size is at least an order-of-magnitude below expectations. We apply electron microscopy and resonance mass measurement methods to quantify the size and uniformity of the nanobubbles, and probe the dependence of the critical bubble diameter on the lipid concentration. We anticipate that further analysis of this unexpected microbubble sudden contraction regime can lead to more robust technologies for making monodisperse nanobubbles.

Ultrasound-guided transfection of claudin-5 improves lung endothelial barrier function in lung injury without impairing innate immunity.

In acute lung injury, the lung endothelial barrier is compromised. Loss of endothelial barrier integrity occurs in association with decreased levels of the tight junction protein claudin-5. Restoration of their levels by gene transfection may improve the vascular barrier, but how to limit transfection solely to regions of the lung that are injured is unknown. We hypothesized that thoracic ultrasound in combination with intravenous microbubbles (USMBs) could be used to achieve regional gene transfection in injured lung regions and improve endothelial barrier function. Since air blocks ultrasound energy, insonation of the lung is only achieved in areas of lung injury (edema and atelectasis); healthy lung is spared. Cavitation of the microbubbles achieves local tissue transfection. Here we demonstrate successful USMB-mediated gene transfection in the injured lungs of mice. After thoracic insonation, transfection was confined to the lung and only occurred in the setting of injured (but not healthy) lung. In a mouse model of acute lung injury, we observed downregulation of endogenous claudin-5 and an acute improvement in lung vascular leakage and in oxygenation after claudin-5 overexpression by transfection. The improvement occurred without any impairment of the immune response as measured by pathogen clearance, alveolar cytokines, and lung histology. In conclusion, USMB-mediated transfection targets injured lung regions and is a novel approach to the treatment of lung injury.NEW & NOTEWORTHY Acute respiratory distress syndrome is characterized by spatial heterogeneity, with severely injured lung regions adjacent to relatively normal areas. This makes targeting treatment to the injured regions difficult. Here we use thoracic ultrasound and intravenous microbubbles (USMBs) to direct gene transfection specifically to injured lung regions. Transfection of the tight junction protein claudin-5 improved oxygenation and decreased vascular leakage without impairing innate immunity. These findings suggest that USMB is a novel treatment for ARDS.

Radioenhancement with the Combination of Docetaxel and Ultrasound Microbubbles: In Vivo Prostate Cancer.

Using an in vitro prostate cancer model, we previously demonstrated the significant enhancement of radiotherapy (XRT) with the combined treatment of docetaxel (Taxotere; TXT) and ultrasound-microbubbles (USMB). Here, we extend these findings to an in vivo cancer model. Severe combined immune-deficient male mice were xenografted with the PC-3 prostate cancer cell line in the hind leg and treated with USMB, TXT, radiotherapy (XRT), and their combinations. The tumors were imaged with ultrasound pre-treatment and 24 h post-treatment, following which they were extracted for the histological analysis of the tumor-cell death (DN; H&E) and apoptosis (DA; TUNEL). The tumors' growths were assessed for up to ~6 weeks and analysed using the exponential Malthusian tumor-growth model. The tumors' doubling time (VT) was characterized as growth (positive) or shrinkage (negative). The cellular death and apoptosis increased ~5-fold with the TXT + USMB + XRT (Dn = 83% and Da = 71%) compared to the XRT alone (Dn = 16% and Da = 14%), and by ~2-3-fold with the TXT + XRT (Dn = 50% and Da = 38%) and USMB + XRT (Dn = 45% and Da = 27%) compared to the XRT. The USMB enhanced the cellular bioeffects of the TXT by ~2-5-fold with the TXT + USMB (Dn = 42% and Da = 50%), compared with the TXT alone (Dn = 19% and Da = 9%). The USMB alone caused cell death (Dn = 17% and Da = 10%) compared to the untreated control (Dn = 0.4% and Da = 0%). The histological cellular bioeffects were correlated with the changes in the ultrasound RF mid-band-fit data, which were associated with the cellular morphology. The linear regression analysis displayed a positive linear correlation between the mid-band fit and the overall cell death (R2 = 0.9164), as well as a positive linear correlation between the mid-band fit and the apoptosis (R2 = 0.8530). These results demonstrate a correlation between the histological and spectral measurements of the tissue microstructure and that cellular morphological changes can be detected by ultrasound scattering analysis. In addition, the tumor volumes from the triple-combination treatment were significantly smaller than those from the control, XRT, USMB + XRT, and TXT + XRT, from day 2 onward. The TXT + USMB + XRT-treated tumors shrank from day 2 and at each subsequent time-point measured (VT ~-6 days). The growth of the XRT-treated tumors was inhibited during the first 16 days, following which the tumors grew (VT ~9 days). The TXT + XRT and USMB + XRT groups displayed an initial decrease in tumor size (day 1-14; TXT + XRT VT ~-12 days; USMB + XRT VT ~-33 days), followed by a growth phase (day 15-37; TXT + XRT VT ~11 days; USMB + XRT VT ~22 days). The triple-combination therapy induced tumor shrinkage to a greater extent than any of the other treatments. This study demonstrates the in vivo radioenhancement potential of chemotherapy combined with therapeutic ultrasound-microbubble treatment in inducing cell death and apoptosis, as well as long-term tumor shrinkage.

AMPK is required for recovery from metabolic stress induced by ultrasound microbubble treatment.

Ultrasound-stimulated microbubble (USMB) treatment is a promising strategy for cancer therapy. USMB promotes drug delivery by sonoporation and enhanced endocytosis, and also impairs cell viability. However, USMB elicits heterogeneous effects on cell viability, with apparently minimal effects on a subset of cells. This suggests that mechanisms of adaptation following USMB allow some cells to survive and/or proliferate. Herein, we used several triple negative breast cancer cells to identify the molecular mechanisms of adaptation to USMB-induced stress. We found that USMB alters steady-state levels of amino acids, glycolytic intermediates, and citric acid cycle intermediates, suggesting that USMB imposes metabolic stress on cells. USMB treatment acutely reduces ATP levels and stimulates the phosphorylation and activation of AMP-activated protein kinase (AMPK). AMPK is required to restore ATP levels and support cell proliferation post-USMB treatment. These results suggest that AMPK and metabolic perturbations are likely determinants of the antineoplastic efficacy of USMB treatment.

Structure-rheology relationship in monoolein liquid crystals.

HYPOTHESES: Monoolein liquid crystals find use in foods and pharmaceuticals. Our hypotheses were: (a) liquid crystal symmetry dominates yielding and large deformation, and (b) strain rate frequency superposition (SRFS) may be used to determine mesophase long and short relaxation times. EXPERIMENTS: Liquid crystal microstructure and rheology were characterised as a function of temperature and composition. Their structure was assessed using small angle X-ray scattering (SAXS) and polarised light microscopy. Small and large deformation rheology was characterised using frequency and amplitude sweeps, large amplitude oscillatory shear tests, and SRFS. FINDINGS: We have contributed to the structure-rheology relationship governing the properties of the lamellar, cubic, and hexagonal mesophases. Initially, we characterised a number of monoolein-water binary phase transitions, which showed similar behaviour with earlier efforts. Frequency sweeps revealed that the cubic phases had the highest elasticity followed by the lamellar and hexagonal phases. The stiffening and thickening ratios, extracted from the Lissajous-Bowditch plots, were used to quantify intra-cycle non-linearities. The cubic phases displayed abrupt yielding with more pronounced stiffening and thinning behaviour compared to the others. Each liquid crystal phase displayed unique rheological behaviour upon large deformation and, by linking rheology with SAXS and composition, we show that their symmetry defined their rheology.

Controlled Shrinkage of Microfluidically Generated Microbubbles by Tuning Lipid Concentration.

Monodisperse microbubbles with diameters less than 10 µm are desirable in several ultrasound imaging and therapeutic delivery applications. However, conventional approaches to synthesize microbubbles, which are usually agitation-based, produce polydisperse bubbles that are less desirable because of their heterogeneous response when exposed to an ultrasound field. Microfluidics technology has the unique advantage of generating size-controlled monodisperse microbubbles, and it is now well established that the diameter of microfluidically made microbubbles can be tuned by varying the liquid flow rate, gas pressure, and dimensions of the microfluidic channel. It is also observed that once the microbubbles form, the bubbles shrink and eventually stabilize to a quasi-equilibrium diameter, and that the rate of stabilization is related to the lipid solution. However, how the lipid solution concentration affects the degree of bubble shrinkage, and the stable size of microbubbles, has not been thoroughly examined. Here, we investigate whether and how the lipid concentration affects the degree of microbubble shrinkage. Namely, we utilize a flow-focusing microfluidic geometry to generate monodisperse bubbles, and observe the effect of gas composition (2.5, 1.42, and 0.17 wt % octafluoropropane in nitrogen) and lipid concentration (1-16 mg/mL) on the degree of microbubble shrinkage. For the lipid system and gas utilized in these experiments, we observe a monotonic increase in the degree of microbubble shrinkage with decreasing lipid concentration, and no dependency on the gas composition. We hypothesize that the degree of shrinkage is related to lipid concentration by the self-assembly of lipids on the gas-liquid interface during bubble generation and subsequent lipid packing on the interface during shrinkage, which is arrested when a maximum packing density is achieved. We anticipate that this approach for creating and tuning the size of monodisperse microbubbles will find utility in biomedical applications, such as contrast-enhanced ultrasound imaging and ultrasound-triggered gene delivery.

Synergistic enhancement of cell death by triple combination therapy of docetaxel, ultrasound and microbubbles, and radiotherapy on PC3 a prostate cancer cell line.

The application of ultrasound and microbubbles (USMB) has been shown to enhance both chemotherapy and radiotherapy. This study investigated the potential of triple combination therapy comprised of USMB, docetaxel (Taxotere: TXT) chemotherapy and XRT to enhance treatment efficacy. Prostate cancer (PC3) cells in suspension were treated with various combinations of USMB, chemotherapy and radiotherapy. Cells were treated with ultrasound and microbubbles (500 kHz pulse center frequency, 580 kPa peak negative pressure, 10 µs pulse duration, 60 s insonation time and 2% Definity microbubbles (v/v)), XRT (2 Gy), and Taxotere (TXT) at concentrations ranging from 0.001 to 0.1 nM for 5- and 120-minutes duration. Following treatment, cell viability was assessed using a clonogenic assay. Therapeutic efficiency of the combined treatments depended on chemotherapy and microbubble exposure conditions. Under the exposure conditions of the study, the triple combination therapy synergistically enhanced clonogenic cell death compared to single and double combination therapy. Cell viability of ∼2% was achieved with the triple combination therapy corresponding to ∼29, ∼37, and ∼38 folds decrease compared to XRT (57%), USMB (74%) and TXT (76%) alone conditions, respectively. In addition, the triple combination therapy decreased cell viability by ∼29, ∼19- and ∼11 folds compared to TXT2hr + USMB (58%), TXT2hr + XRT (37%), and USMB + XRT (22%), respectively. The in vivo PC3 tumours showed that USMB significantly enhanced cell death through detection of apoptosis (TUNEL) with both TXT and TXT + XRT. The study demonstrated that the triple combination therapy can significantly enhance cell death in prostate cancer cells both in vitro and in vivo under relatively low chemotherapy and ionizing radiation doses.

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease.

The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cell-based experimental assessment reveals several clinically-relevant drug repurposing candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

Targeted enhancement of flotillin-dependent endocytosis augments cellular uptake and impact of cytotoxic drugs.

Cellular uptake is limiting for the efficacy of many cytotoxic drugs used to treat cancer. Identifying endocytic mechanisms that can be modulated with targeted, clinically-relevant interventions is important to enhance the efficacy of various cancer drugs. We identify that flotillin-dependent endocytosis can be targeted and upregulated by ultrasound and microbubble (USMB) treatments to enhance uptake and efficacy of cancer drugs such as cisplatin. USMB involves targeted ultrasound following administration of encapsulated microbubbles, used clinically for enhanced ultrasound image contrast. USMB treatments robustly enhanced internalization of the molecular scaffold protein flotillin, as well as flotillin-dependent fluid-phase internalization, a phenomenon dependent on the protein palmitoyltransferase DHHC5 and the Src-family kinase Fyn. USMB treatment enhanced DNA damage and cell killing elicited by the cytotoxic agent cisplatin in a flotillin-dependent manner. Thus, flotillin-dependent endocytosis can be modulated by clinically-relevant USMB treatments to enhance drug uptake and efficacy, revealing an important new strategy for targeted drug delivery for cancer treatment.

Analysis of the cytotoxic effects of combined ultrasound, microbubble and nucleoside analog combinations on pancreatic cells in vitro.

Ultrasonically-stimulated microbubbles enhance the therapeutic effects of various chemotherapy drugs. However, the application of ultrasound and microbubbles (USMB) for enhancing the therapeutic effect of nucleoside analogs, which are used as front-line treatments in a range of cancers, and its underlying mechanism is not well understood. This study investigated the effect of gemcitabine, a nucleoside analog drug, in combination with USMB in increasing cell cytotoxicity relative to either treatment alone in BxPC3 pancreatic cancer cells. Cells were sonicated using low frequency (0.5 MHz) ultrasound in combination with Definity® microbubbles (1.7% v/v) in the presence of 1 µM of gemcitabine for a total of 2 h. USMB in combination with gemcitabine decreased cell viability (48 h) to 44.7 ±â€¯5.2%, 27.7 ±â€¯4.3%, and 12.5 ±â€¯3.4% with increasing ultrasound peak negative pressures (220, 360, 530 kPa) from 84.7 ±â€¯3.6%, 54.2 ±â€¯3.8%, and 26.8 ±â€¯3.0%, respectively, when USMB was applied in the absence of drug. We further confirmed that USMB did not enhance the internalization of 1 µM of a radiolabeled nucleoside analog (2-chloroadenosine) at each of the three chosen ultrasound PNPs, determined by radiolabeled scintillation counting. These data suggest that USMB in combination with nucleoside analog drugs leads to an additive effect on cell toxicity and that USMB does not impair transporter-mediated uptake of nucleoside analogs.

Ultrasound and microbubble induced release from intracellular compartments.

BACKGROUND: Ultrasound and microbubbles (USMB) have been shown to enhance the intracellular uptake of molecules, generally thought to occur as a result of sonoporation. The underlying mechanism associated with USMB-enhanced intracellular uptake such as membrane disruption and endocytosis may also be associated with USMB-induced release of cellular materials to the extracellular milieu. This study investigates USMB effects on the molecular release from cells through membrane-disruption and exocytosis. RESULTS: USMB induced the release of 19% and 67% of GFP from the cytoplasm in viable and non-viable cells, respectively. Tfn release from early/recycling endosomes increased by 23% in viable cells upon USMB treatment. In addition, the MFI of LAMP-1 antibody increased by 50% in viable cells, suggesting USMB-stimulated lysosome exocytosis. In non-viable cells, labeling of LAMP-1 intracellular structures in the absence of cell permeabilization by detergents suggests that USMB-induced cell death correlates with lysosomal permeabilization. CONCLUSIONS: In conclusion, USMB enhanced the molecular release from the cytoplasm, lysosomes, and early/recycling endosomes.

Honey, I shrunk the bubbles: microfluidic vacuum shrinkage of lipid-stabilized microbubbles.

We present a microfluidic technique that shrinks lipid-stabilized microbubbles from O(100) to O(1) µm in diameter - the size that is desirable in applications as ultrasound contrast agents. We achieve microbubble shrinkage by utilizing vacuum channels that are adjacent to the microfluidic flow channels to extract air from the microbubbles. We tune a single parameter, the vacuum pressure, to accurately control the final microbubble size. Finally, we demonstrate that the resulting O(1) µm diameter microbubbles have similar stability to microfluidically generated microbubbles that are not exposed to vacuum shrinkage. We anticipate that, with additional scale-up, this simple approach to shrink microbubbles generated microfluidically will be desirable in ultrasound imaging and therapeutic applications.

Microvascular contrast enhancement in optical coherence tomography using microbubbles.

Gas microbubbles (MBs) are investigated as intravascular optical coherence tomography (OCT) contrast agents. Agar + intralipid scattering tissue phantoms with two embedded microtubes were fabricated to model vascular blood flow. One was filled with human blood, and the other with a mixture of human blood + MB. Swept-source structural and speckle variance (sv) OCT images, as well as speckle decorrelation times, were evaluated under both no-flow and varying flow conditions. Faster decorrelation times and higher structural and svOCT image contrasts were detected in the presence of MB in all experiments. The effects were largest in the svOCT imaging mode, and uniformly diminished with increasing flow velocity. These findings suggest the feasibility of utilizing MB for tissue hemodynamic investigations and for microvasculature contrast enhancement in OCT angiography.

Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms.

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

USMB-induced synergistic enhancement of aminoglycoside antibiotics in biofilms.

This study evaluated the effect of combining antibiotics with ultrasound and microbubbles (USMB) toward the eradication of biofilms. Pseudomonas aeruginosa PAO1 biofilms were treated with the antibiotics gentamicin sulfate or streptomycin sulfate, or a combination of USMB with the respective antibiotics. Biofilm structure was quantified using confocal laser scanning microscopy with COMSTAT analysis, while activity was measured as whole-biofilm CO2 production in a continuous-flow biofilm model. The combined antibiotic-USMB treatment significantly impacted biofilm biomass, thickness and surface roughness compared to antibiotics alone (p<0.05). USMB exposure caused the formation of craters (5-20µm in diameter) in the biofilms, and when combined with gentamicin, activity was significantly lower, compared to gentamicin, USMB or untreated controls, respectively. Interestingly, the CO2 production rate following combined streptomycin-USMB treatment was higher than after streptomycin alone, but significantly lower than USMB alone and untreated control. These results show strong evidence of a synergistic effect between antibiotics and USMB, although the varied response to different antibiotics emphasize the need to optimize the USMB exposure conditions to maximize this synergism and ultimately transfer this technology into clinical or industrial practice.

Enhancing laser therapy using PEGylated gold nanoparticles combined with ultrasound and microbubbles.

BACKGROUND: Gold nanorod (AuNR) laser therapy (LT) is a non-invasive method of increasing the temperature of a target tissue using near infrared light. In this study, the effects of ultrasound and microbubbles (USMB) with AuNR and LT were investigated on cell viability. METHODS: MDA-MB-231 cells in suspension were treated with three different treatment combinations of AuNR, LT and USMB (Pneg=0.6 or 1.0 MPa): (1) AuNR with USMB followed by LT, (2) AuNR and LT followed by USMB, and (3) USMB followed by AuNR and LT. Cells were also exposed to USMB and LT without AuNR. The USMB conditions were: 500 kHz frequency, 16 cycles, 1kHz pulse repetition frequency for 1 min in the presence of Definity microbubbles (1.7% v/v). AuNR and LT conditions were: mPEG coated AuNR at 3×10(11) np/mL and 1.9 W/cm(2) for 3 min. Following the treatment, cell viability was assessed using propidium iodide (PI) fluorescent marker and flow cytometry (VPI), and colony assay (VCA). Cell viabilities were compared using a non-parametric Mann-Whitney U-test and synergism was assessed using the Bliss Independence Model. RESULTS AND DISCUSSION: USMB improved cell death when combined with AuNR and LT. VPI of 17±2% (at 0.6 MPa) and 11±4% (at 1.0 MPa) were observed with combined treatment of AuNR and USMB followed by LT compared to VPI of 60±2% (at 0.6 MPa) and 42±3% (at 1.0 MPa) with USMB alone and VPI of 22±3% for AuNR and LT. The combined effect of AuNR and LT with USMB was additive regardless of treatment order. VCA results agreed with the additive effect caused by combining AuNR and LT with USMB for all treatment orders. In the absence of AuNR, samples exposed to LT prior to USMB at 0.6 MPa increased VPI by 13% (p<0.01) showing a protective effect. CONCLUSION: Combining AuNR and LT with USMB resulted in an additive effect on cell viability compared to AuNR and LT, or USMB. In addition, cells exposed to low intensity NIR light appear to be protected against USMB exposure.

Quantitative ultrasound characterization of tumor cell death: ultrasound-stimulated microbubbles for radiation enhancement.

The aim of this study was to assess the efficacy of quantitative ultrasound imaging in characterizing cancer cell death caused by enhanced radiation treatments. This investigation focused on developing this ultrasound modality as an imaging-based non-invasive method that can be used to monitor therapeutic ultrasound and radiation effects. High-frequency (25 MHz) ultrasound was used to image tumor responses caused by ultrasound-stimulated microbubbles in combination with radiation. Human prostate xenografts grown in severe combined immunodeficiency (SCID) mice were treated using 8, 80, or 1000 µL/kg of microbubbles stimulated with ultrasound at 250, 570, or 750 kPa, and exposed to 0, 2, or 8 Gy of radiation. Tumors were imaged prior to treatment and 24 hours after treatment. Spectral analysis of images acquired from treated tumors revealed overall increases in ultrasound backscatter intensity and the spectral intercept parameter. The increase in backscatter intensity compared to the control ranged from 1.9±1.6 dB for the clinical imaging dose of microbubbles (8 µL/kg, 250 kPa, 2 Gy) to 7.0±4.1 dB for the most extreme treatment condition (1000 µL/kg, 750 kPa, 8 Gy). In parallel, in situ end-labelling (ISEL) staining, ceramide, and cyclophilin A staining demonstrated increases in cell death due to DNA fragmentation, ceramide-mediated apoptosis, and release of cyclophilin A as a result of cell membrane permeabilization, respectively. Quantitative ultrasound results indicated changes that paralleled increases in cell death observed from histology analyses supporting its use for non-invasive monitoring of cancer treatment outcomes.

In vivo feasibility study of ultrasound potentiated collagenase therapy of chronic total occlusions.

Arterial chronic total occlusions (CTOs) pose considerable challenges for percutaneous interventions, due primarily to the presence of stiff proximal fibrous caps (PFCs) which act as a barrier to the penetration of guide wires. A new approach under development for improving the success rate of guide wire crossing in CTOs is to employ collagenase to degrade the mechanical integrity of the PFCs. This has been shown to be feasible in preclinical work and in a Phase 1 clinical trial. In a recent study we demonstrated using ex vivo experimental CTO specimens that ultrasound-stimulated microbubbles (USMBs) could potentiate the effects of collagenase and result in increased mechanical degradation of the PFCs of CTOs. Here we report the results of the first in vivo study examining the feasibility of this approach, which demonstrates that the force required to puncture through the PFCs of CTOs is reduced with combined USMB+collagenase treatments relative to collagenase only treatments. This approach has the potential to further improve the efficacy of the emerging technique of collagenase facilitation of percutaneous interventions for CTO.