Recent efforts in the area of acoustic droplet vaporization with the objective of designing extravascular ultrasound contrast agents has led to the development of stabilized, lipid-encapsulated ...nanodroplets of the highly volatile compound decafluorobutane (DFB). We developed two methods of generating DFB droplets, the first of which involves condensing DFB gas (boiling point from −1.1 to −2 °C) followed by extrusion with a lipid formulation in HEPES buffer. Acoustic droplet vaporization of micrometer-sized lipid-coated droplets at diagnostic ultrasound frequencies and mechanical indices were confirmed optically. In our second formulation methodology, we demonstrate the formulation of submicrometer-sized lipid-coated nanodroplets based upon condensation of preformed microbubbles containing DFB. The droplets are routinely in the 200–300 nm range and yield microbubbles on the order of 1–5 μm once vaporized, consistent with ideal gas law expansion predictions. The simple and effective nature of this methodology allows for the development of a variety of different formulations that can be used for imaging, drug and gene delivery, and therapy. This study is the first to our knowledge to demonstrate both a method of generating ADV agents by microbubble condensation and formulation of primarily submicrometer droplets of decafluorobutane that remain stable at physiological temperatures. Finally, activation of DFB nanodroplets is demonstrated using pressures within the FDA guidelines for diagnostic imaging, which may minimize the potential for bioeffects in humans. This methodology offers a new means of developing extravascular contrast agents for diagnostic and therapeutic applications.
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IJS, KILJ, NUK, PNG, UL, UM
Ultrasonically activated phase-change contrast agents (PCCAs) based on perfluorocarbon droplets have been proposed for a variety of therapeutic and diagnostic clinical applications. When generated at ...the nanoscale, droplets may be small enough to exit the vascular space and then be induced to vaporize with high spatial and temporal specificity by externally-applied ultrasound. The use of acoustical techniques for optimizing ultrasound parameters for given applications can be a significant challenge for nanoscale PCCAs due to the contributions of larger outlier droplets. Similarly, optical techniques can be a challenge due to the sub-micron size of nanodroplet agents and resolution limits of optical microscopy. In this study, an optical method for determining activation thresholds of nanoscale emulsions based on the in vitro distribution of bubbles resulting from vaporization of PCCAs after single, short (<10 cycles) ultrasound pulses is evaluated. Through ultra-high-speed microscopy it is shown that the bubbles produced early in the pulse from vaporized droplets are strongly affected by subsequent cycles of the vaporization pulse, and these effects increase with pulse length. Results show that decafluorobutane nanoemulsions with peak diameters on the order of 200 nm can be optimally vaporized with short pulses using pressures amenable to clinical diagnostic ultrasound machines.
Breast cancer is a diverse and complex disease that remains one of the leading causes of death among women. Novel, outside-of-the-box imaging and treatment methods are needed to supplement currently ...available technologies. In this study, we present evidence for the intracellular delivery and ultrasound-stimulated activation of folate receptor (FR)-targeted phase-change contrast agents (PCCAs) in MDA-MB-231 and MCF-7 breast cancer cells in vitro. PCCAs are lipid-coated, perfluorocarbon-filled particles formulated as nanoscale liquid droplets capable of vaporization into gaseous microbubbles for imaging or therapy. Cells were incubated with 1:1 decafluorobutane (DFB)/octafluoropropane (OFP) PCCAs for 1h, imaged via confocal microscopy, exposed to ultrasound (9MHz, MI=1.0 or 1.5), and imaged again after insonation. FR-targeted PCCAs were observed intracellularly in both cell lines, but uptake was significantly greater (p<0.001) in MDA-MB-231 cells (93.0% internalization at MI=1.0, 79.5% at MI=1.5) than MCF-7 cells (42.4% internalization at MI=1.0, 35.7% at MI=1.5). Folate incorporation increased the frequency of intracellular PCCA detection 45-fold for MDA-MB-231 cells and 7-fold for MCF-7 cells, relative to untargeted PCCAs. Intracellularly activated PCCAs ranged from 500nm to 6μm (IQR=800nm–1.5μm) with a mean diameter of 1.15±0.59 (SD) microns. The work presented herein demonstrates the feasibility of PCCA intracellular delivery and activation using breast cancer cells, illuminating a new platform toward intracellular imaging or therapeutic delivery with ultrasound.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Currently available microbubbles used for ultrasound imaging and therapeutics are limited to intravascular space due to their size distribution in the micron range. Phase-change contrast agents ...(PCCAs) have been proposed as a means to overcome this limitation, since droplets formed in the hundred nanometer size range might be able to extravasate through leaky microvasculature, after which they could be activated to form larger highly echogenic microbubbles. Existing PCCAs in the sub-micron size range require substantial acoustic energy to be vaporized, increasing the likelihood of unwanted bioeffects. Thus, there exists a need for PCCAs with reduced acoustic activation energies for use in imaging studies. In this article, it is shown that decafluorobutane, which is normally a gas at room temperature, can be incorporated into metastable liquid sub-micron droplets with appropriate encapsulation methods. The resulting droplets are activatable with substantially less energy than other favored PCCA compounds. Decafluorobutane nanodroplets may present a new means to safely extend ultrasound imaging beyond the vascular space.
Phase-change contrast agents (PCCAs) provide a dynamic platform to approach problems in medical ultrasound (US). Upon US-mediated activation, the liquid core vaporizes and expands to produce a gas ...bubble ideal for US imaging and therapy. In this study, we demonstrate through high-speed video microscopy and US interrogation that PCCAs composed of highly volatile perfluorocarbons (PFCs) exhibit unique acoustic behavior that can be detected and differentiated from standard microbubble contrast agents. Experimental results show that when activated with short pulses PCCAs will over-expand and undergo unforced radial oscillation while settling to a final bubble diameter. The size-dependent oscillation phenomenon generates a unique acoustic signal that can be passively detected in both time and frequency domain using confocal piston transducers with an 'activate high' (8 MHz, 2 cycles), 'listen low' (1 MHz) scheme. Results show that the magnitude of the acoustic 'signature' increases as PFC boiling point decreases. By using a band-limited spectral processing technique, the droplet signals can be isolated from controls and used to build experimental relationships between concentration and vaporization pressure. The techniques shown here may be useful for physical studies as well as development of droplet-specific imaging techniques.
Recent efforts using perfluorocarbon (PFC) nanoparticles in conjunction with acoustic droplet vaporization has introduced the possibility of expanding the diagnostic and therapeutic capability of ...ultrasound contrast agents to beyond the vascular space. Our laboratories have developed phase-change nanoparticles (PCNs) from the highly volatile PFCs decafluorobutane (DFB, bp =-2 °C) and octafluoropropane (OFP, bp =-37 °C ) for acoustic droplet vaporization. Studies with commonly used clinical ultrasound scanners have demonstrated the ability to vaporize PCN emulsions with frequencies and mechanical indices that may significantly decrease tissue bioeffects. In addition, these contrast agents can be formulated to be stable at physiological temperatures and the perfluorocarbons can be mixed to modulate the balance between sensitivity to ultrasound and general stability. We herein discuss our recent efforts to develop finely-tuned diagnostic/molecular imaging agents for tissue interrogation. We discuss studies currently under investigation as well as potential diagnostic and therapeutic paradigms that may emerge as a result of formulating PCNs with low boiling point PFCs.
Continued advances in the field of ultrasound and ultrasound contrast agents have created new approaches to imaging and medical intervention. Phase-shift perfluorocarbon droplets, which can be ...vaporized by ultrasound energy to transition from the liquid to the vapor state, are one of the most highly researched alternatives to clinical ultrasound contrast agents (i.e., microbubbles). In this paper, part of a special issue on methods in biomedical ultrasonics, we survey current techniques to prepare ultrasound-activated nanoscale phase-shift perfluorocarbon droplets, including sonication, extrusion, homogenization, microfluidics, and microbubble condensation. We provide example protocols and discuss advantages and limitations of each approach. Finally, we discuss best practice in characterization of this class of contrast agents with respect to size distribution and ultrasound activation.
Phase-change contrast agents (PCCAs) are perfluorocarbon nanodroplets (NDs) that have been widely studied for ultrasound imaging in vitro, pre-clinical studies, and most recently incorporated a ...variant of PCCAs, namely a microbubble-conjugated microdroplet emulsion, into the first clinical studies. Their properties also make them attractive candidates for a variety of diagnostic and therapeutic applications including drug-delivery, diagnosis and treatment of cancerous and inflammatory diseases, as well as tumor-growth tracking. However, control over the thermal and acoustic stability of PCCAs both in vivo and in vitro has remained a challenge for expanding the potential utility of these agents in novel clinical applications. As such, our objective was to determine the stabilizing effects of layer-by-layer assemblies and its effect on both thermal and acoustic stability.
We utilized layer-by-layer (LBL) assemblies to coat the outer PCCA membrane and characterized layering by measuring zeta potential and particle size. Stability studies were conducted by; 1) incubating the LBL-PCCAs at atmospheric pressure at 37
C and 45
C followed by; 2) ultrasound-mediated activation at 7.24 MHz and peak-negative pressures ranging from 0.71 - 5.48 MPa to ascertain nanodroplet activation and resultant microbubble persistence. The thermal and acoustic properties of decafluorobutane gas-condensed nanodroplets (DFB-NDs) layered with 6 and 10 layers of charge-alternating biopolymers, (LBL
NDs and LBL
NDs) respectively, were studied and compared to non-layered DFB-NDs. Half-life determinations were conducted at both 37
C and 45
C with acoustic droplet vaporization (ADV) measurements occurring at 23
C.
Successful application of up to 10 layers of alternating positive and negatively charged biopolymers onto the surface membrane of DFB-NDs was demonstrated. Two major claims were substantiated in this study; namely, (1) biopolymeric layering of DFB-NDs imparts a thermal stability up to an extent; and, (2) both LBL
NDs and LBL
NDs did not appear to alter particle acoustic vaporization thresholds, suggesting that the thermal stability of the particle may not necessarily be coupled with particle acoustic vaporization thresholds.
Results demonstrate that the layered PCCAs had higher thermal stability, where the half-lifes of the LBL
NDs are significantly increased after incubation at 37
C and 45
C. Furthermore, the acoustic vaporization profiles the DFB-NDs, LBL
NDs, and LBL
NDs show that there is no statistically significant difference between the acoustic vaporization energy required to initiate acoustic droplet vaporization.
•First identification of a prospective binding pocket in the Ku70/80 heterodimer.•First identification of a small molecule inhibitor of the Ku70/80 heterodimer.•Inhibitor inhibits key elements of DNA ...double strand break repair.•Inhibitor sensitizes human cell lines to ionizing radiation.•Inhibitor can be a template for design of radiation- or chemo-sensitizing drugs.
Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Ultrasound contrast agents are known to enhance high intensity focused ultrasound (HIFU) ablation, but these perfluorocarbon microbubbles are limited to the vasculature, have a short half-life in ...vivo, and may result in unintended heating away from the target site. Herein, a nano-sized (100-300 nm), dual perfluorocarbon (decafluorobutane/dodecafluoropentane) droplet that is stable, is sufficiently small to extravasate, and is convertible to micron-sized bubbles upon acoustic activation was investigated. Microbubbles and nanodroplets were incorporated into tissue-mimicking acrylamide-albumin phantoms. Microbubbles or nanodroplets at 0.1 × 10(6) per cm(3) resulted in mean lesion volumes of 80.4 ± 33.1 mm(3) and 52.8 ± 14.2 mm(3) (mean ± s.e.), respectively, after 20 s of continuous 1 MHz HIFU at a peak negative pressure of 4 MPa, compared to a lesion volume of 1.0 ± 0.8 mm(3) in agent-free control phantoms. Magnetic resonance thermometry mapping during HIFU confirmed undesired surface heating in phantoms containing microbubbles, whereas heating occurred at the acoustic focus of phantoms containing the nanodroplets. Maximal change in temperature at the target site was enhanced by 16.9% and 37.0% by microbubbles and nanodroplets, respectively. This perfluorocarbon nanodroplet has the potential to reduce the time to ablate tumors by one-third during focused ultrasound surgery while also safely enhancing thermal deposition at the target site.
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