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Radiation therapy (RT) causes DNA damage through ionization, leading to double-strand breaks. In addition, it generates reactive oxygen species (ROS), which are toxic to tumor cells and the vasculature. However, hypoxic regions in the tumor have been shown to not only decrease treatment response but also increase the likelihood of recurrence and metastasis. Ultrasound-sensitive micro-bubbles are emerging as a useful diagnostic and therapeutic tool within RT. Contrast-enhanced ultrasound (CEUS) has shown great promise in early prediction of tumor response to RT. Ultrasound-triggered micro-bubble cavitation has also been shown to induce bio-effects that can sensitize angiogenic tumor vessels to RT. Additionally, ultrasound can trigger the release of drugs from micro-bubble carriers via localized micro-bubble destruction. This approach has numerous applications in RT, including targeted oxygen delivery before radiotherapy. Furthermore, micro-bubbles can be used to locally create ROS without radiation. Sonodynamic therapy uses focused ultrasound and a sonosensitizer to selectively produce ROS in the tumor region and has been explored as a treatment option for cancer. This review summarizes emerging applications of ultrasound contrast agents in RT and ROS augmentation.

Background and purpose: Lonidamine is a hexokinase II inhibitor, works as an anticancer molecule, and is extensively explored in clinical trials. Limited information prevails about the stability-indicating methods which could determine the forced degradation of lonidamine under stressed conditions. Hence, we report the use of a rapid, sensitive, reproducible, and highly accurate liquid chromatography and mass spectrometry method to analyze lonidamine degradation. Experimental approach: The Xbridge BEH shield reverse phase C18 column (2.5 µm, 4.6 × 75 mm) using isocratic 50:50 water: acetonitrile with 0.1% formic acid can detect lonidamine with help of mass spectrometer in tandem with an ultraviolet (UV) detector at 260 nm wavelength. Findings/ Results: A linear curve with r² > 0.99 was obtained for tandem liquid chromatography-mass spectrometry (LC-MS)-UV based detections. This study demonstrated (in the present set up of isocratic elution) that LC-MS based detection has a relatively high sensitivity (S/N (10 ng/mL): 220 and S/N (20 ng/mL): 945) and accuracy at lower detection and quantitation levels, respectively. In addition to developing the LC-MS method, we also report that the current method is stability-indicating and shows that lonidamine gets degraded over time under all three stress conditions; acidic, basic, and oxidative. Conclusion and implications: LC-MS based quantitation of lonidamine proved to be a better method compared to high-performance liquid chromatography (HPLC)-UV detections for mapping lonidamine degradation. This is the first report on the stability-indicating method for studying the forced degradation of lonidamine using LC-MS method..

Prior work has shown that microbubble-assisted delivery of oxygen improves tumor oxygenation and radiosensitivity, albeit over a limited duration. Lonidamine (LND) has been investigated because of its ability to stimulate glycolysis, lactate production, inhibit mitochondrial respiration, and inhibit oxygen consumption rates in tumors but suffers from poor bioavailability. The goal of this work was to characterize LND-loaded oxygen microbubbles and assess their ability to oxygenate a human head and neck squamous cell carcinoma (HNSCC) tumor model, while also assessing LND biodistribution. In tumors treated with surfactant-shelled microbubbles with oxygen core (SE61O(2)) and ultrasound, pO(2) levels increased to a peak 19.5 ± 9.7 mmHg, 50 s after injection and returning to baseline after 120 s. In comparison, in tumors treated with SE61O(2)/LND and ultrasound, pO(2) levels showed a peak increase of 29.0 ± 8.3 mmHg, which was achieved 70 s after injection returning to baseline after 300 s (p < 0.001). The co-delivery of O(2) and LND via SE61 also showed an improvement of LND biodistribution in both plasma and tumor tissues (p < 0.001). In summary, ultrasound-sensitive microbubbles loaded with O(2) and LND provided prolonged oxygenation relative to oxygenated microbubbles alone, as well as provided an ability to locally deliver LND, making them more appropriate for clinical translation.

Ultrasound contrast agents consist of stabilized microbubbles. We are developing a surfactant-stabilized microbubble platform with a shell composed of Span 60 (Sorbitan monostearate) and an emulsifying agent, water-soluble vitamin E (α-tocopheryl poly(ethylene glycol) succinate, abbreviated as TPGS), named SE61. The microbubbles act both as an imaging agent and a vehicle for delivering oxygen to hypoxic areas in tumors. For clinical use, it is important that a platform be stable under storage at room temperature. To accomplish this, a majority of biologicals are prepared as freeze-dried powders, which also eliminates the necessity of a cold chain. The interfaces among the surfactants, gas, and liquids are subject to disruption in both the freezing and drying phases. Using thermocouples to monitor temperature profiles, differential scanning calorimetry to determine the phase transitions, and acoustic properties to gauge the degree of microbubble disruption, the effects of the freezing rate and the addition of different concentrations of lyoprotectants were determined. Slower cooling rates achieved by freezing the samples in a −20 °C bath were found to be reproducible and produce contrast agents with acceptable acoustical properties. The ionic strength of the solutions and the concentration of the lyoprotectant determined the glass-transition temperature (T(g)′) of the frozen sample, which determines at what temperature samples can be dried without collapse. Crucially, we found that the shelf stability of surfactant-shelled oxygen microbubbles can be enhanced by increasing the lyoprotectant (glucose) concentration from 1.8 to 5.0% (w/v), which prevents the melt temperature (T(m)) of the TPGS phase from rising above room temperature. The increase in glucose concentration results in a lowering of T(m) of the emulsifying agent, preventing a phase change in the liquid-crystalline phase and allowing for more stable bubbles. We believe that preventing this phase change is necessary to producing stabilized freeze-dried microbubbles.

Ultrasound imaging presents many positive attributes, including safety, real-time imaging, universal accessibility, and cost. However, inherent difficulties in discrimination between soft tissues and tumors prompted development of stabilized microbubble contrast agents. This presents the opportunity to develop agents in which drug is entrapped in the microbubble shell. We describe preparation and characterization of theranostic poly(lactide) (PLA) and pegylated PLA (PEG-PLA) shelled microbubbles that entrap gemcitabine, a commonly used drug for pancreatic cancer (PDAC). Entrapping 6 wt% gemcitabine did not significantly affect drug activity, microbubble morphology, or ultrasound contrast activity compared with unmodified microbubbles. In vitro microbubble concentrations yielding ≥ 500nM entrapped gemcitabine were needed for complete cell death in MIA PaCa-2 PDAC drug sensitivity assays, compared with 62.5 nM free gemcitabine. In vivo administration of gemcitabine-loaded microbubbles to xenograft MIA PaCa-2 PDAC tumors in athymic mice was well tolerated and provided substantial tumoral image enhancement before and after destructive ultrasound pulses. However, no significant differences in tumor growth were observed among treatment groups, in keeping with the in vitro observation that much higher doses of gemcitabine are required to mirror free gemcitabine activity. Statement of significance The preliminary results shown here are encouraging and support further investigation into increased gemcitabine loading. Encapsulation of gemcitabine within polylactic acid (PLA) microbubbles does not damage its activity towards pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) cells. Excellent imaging and evidence of penetration into the highly desmoplastic PDAC tumors is demonstrated. Microbubble destruction was confirmed in vivo, showing that elevated mechanical index shatters the microbubbles for enhanced delivery. The potential to slow PDAC growth in vivo is shown, but higher gemcitabine concentrations are required. Current efforts are directed at increasing drug loading by inclusion of drug-carrying nanoparticles for effective in vivo treatment.

Lonidamine (LND) has been widely explored due to its ability to target metabolic glycolysis and mitochondrial respiration in tumors [4]. However, clinical translation has stalled due to poor bioavailability after oral administration. In this study, we explored three incubation time points of loading oxygen-filled microbubbles with LND (SE61O2-LND) for radiotherapy sensitization by modifying our published methods for creating surfactant-shelled oxygen microbubbles (SE61 O2). Acoustic enhancement and stability were quantified in vitro in a continuously insonated sample holder using a 5 MHz single element transducer which generated a peak positive pressure of 0.69 MPa and a peak negative pressure of 0.25 MP at a PRF of 100 Hz and in a pulsatile flow phantom insonated at a single point using a C1-6 transducer on a GE Logiq E9 system (GE Healthcare) operating at 4.0 MHz at a mechanical index of 0.12. Flow cytometry was performed to analyze the total bubble count for each incubation time point. Lonidamine encapsulation was determined using liquid chromatography-mass spectrometry (LC-MS), the loading approach consisted of incubating the LND and TPGS at 37°C at 0, 24, and 48 hours. Bubbles incubated for 24 hours had an average encapsulation of 2.64 ± 0.32 µg LND/ mL MB, and those incubated for 48 hours had an average of 2.97 ± 0.36 µg LND/ mL MB, while non-incubated micelles had an average of 1.51 ± 0.05 µg LND/ mL MB, both TPGS micelle methods that were incubated showed a statistically significant improvement p < 0.001 compared to non-incubated. This work demonstrates the feasibility of fabricating lonidamine-loaded microbubbles and an improved approach for increasing drug encapsulation. The microbubbles retained their acoustic properties, and by incubating the lonidamine with the TPGS to form micelles, drug loading was increased.

Much of the volume of solid tumors typically exists in a chronically hypoxic microenvironment that has been shown to result in both chemotherapy and radiation therapy resistance. The purpose of this study was to use localized microbubble delivery to overcome hypoxia prior to therapy.In this study, surfactant-shelled oxygen microbubbles were fabricated and injected intravenously to locally elevate tumor oxygen levels when triggered by noninvasive ultrasound in mice with human breast cancer tumors. Changes in oxygen and sensitivity to radiation therapy were then measured.In this work, we show that oxygen-filled microbubbles successfully and consistently increase breast tumor oxygenation levels in a murine model by 20 mmHg, significantly more than control injections of saline solution or untriggered oxygen microbubbles (P .001). Using photoacoustic imaging, we also show that oxygen delivery is independent of hemoglobin transport, enabling oxygen delivery to avascular regions of the tumor. Finally, we show that overcoming hypoxia by this method immediately prior to radiation therapy nearly triples radiosensitivity. This improvement in radiosensitivity results in roughly 30 days of improved tumor control, providing statistically significant improvements in tumor growth and animal survival (P .03).Our findings demonstrate the potential advantages of ultrasound-triggered oxygen delivery to solid tumors and warrant future efforts into clinical translation of the microbubble platform.

This study represents the first attempt to combine surface TRAIL expression and doxorubicin co-encapsulation in a single drug delivery agent in the form of ultrasound-responsive microbubbles that shatter into fragments, or nanoshards, in an ultrasound beam. We compare customized microbubbles of different polymeric shell compositions, and investigate the effect of both shell composition and incorporation of doxorubicin on action against TRAIL-sensitive MDA-MB-231 and TRAIL-resistant MCF7 human breast adenocarcinoma cells. Ligation of TRAIL only significantly impacted MDA-MB-231 cells predominantly by apoptosis, and had minimal effect on MCF12A (normal control) cells. For all shell types, nanoshards had a greater effect (apoptotic death ranging from approximately 25% for 1 wt % LipidPEG to 50% for 100% PLA), reflecting the greater surface area and larger number of particles that ultrasound generates. Encapsulation of doxorubicin generated necrosis in all cell lines, but PEGylation produced less effective necrosis in all cell lines. Co-encapsulation of doxorubicin within the contrast agent shell increased MDA-MB-231 cell death to approximately 40-80%, representing a marked increase over TRAIL alone, reflecting the dramatic effect of shell composition. Additionally, shells that co-encapsulated TRAIL and doxorubicin resulted in approximately 30-60% death in TRAIL-resistant MCF7 human breast adenocarcinoma cells, compared with little apoptotic response in these cells from shells encapsulating TRAIL alone, demonstrating the sensitization effect of the drug. This work has resulted in production of a library of effective ultrasound-triggered, minimally immunogenic, targeted drug delivery agents for potential use in cancer therapy, and represents a promising multifaceted treatment to better serve the population with solid tumors. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1903-1915, 2018.

We have developed oxygen filled microbubbles, SE61O2, for localized, ultrasound-triggered oxygen delivery to hypoxic tumors prior to radiation therapy. Microbubbles, created by sonication, have a shell composed of D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) and sorbitan monostearate. Preliminary studies in mice with breast tumor xenographs showed that increases in oxygen partial pressure levels lasted less than 3 min, which is insufficient for most clinical applications. Hence, we investigated the potential of incorporating a hydrophobic antiglycolytic drug, modeled with Nile red. A new fabrication method was developed by first creating drug-loaded TPGS micelles. The resulting microbubbles had similar shell compositions, physical size, morphology, and acoustic properties as the original method. However, microbubble yield was more than doubled, resulting in twice the encapsulation efficiency. For the TPGS micelle method these include similar shell compositions (94.4 ± 0.6 % Montane 60), physical size post freeze-drying and reconstitution (1.57 ± 0.42 μm), morphology (spherical), and acoustic properties (maximum enhancement 19.92 ± 0.55 dB). However, microbubble yield was more than doubled, resulting in twice the encapsulation efficiency (up to 10.49 %). We propose that a nonideal mixture is formed when the surfactants are combined by the standard method, resulting in the formation of mixed micelles that are more stable, making microbubble creation more difficult during the sonication step.

The composition of microcapsules designed for drug delivery significantly impacts their properties. Ultrasound contrast agents, consisting of stabilized microbubbles (MBs), have emerged as versatile potential drug delivery vehicles to both image and overcome challenges associated with systemic chemotherapy. In our development of polylactic acid MBs decorated with immune-shielding polyethylene glycol chains, we have shown that the balance between acoustic behavior and immune avoidance was scalable and amenable to two distinct PEGylation methods, either incorporation of 5 wt% PEGylated PLA or insertion of 1 wt% PEGylated lipid (LipidPEG) in the polymeric shell. Here we describe the effects of shell compositions on MB functionalization for use in targeted cancer therapy. We chose tumor necrosis factor-related apoptosis inducing ligand (TRAIL) as the targeting ligand, motivated by the ability to both target cells and selectively induce tumor cell death upon binding. Additionally, the MBs were designed to co-encapsulate the chemotherapeutic doxorubicin (Dox) within the shell that works with TRAIL to sensitize resistant cells. We have previously shown that the MBs shatter in response to ultrasound focused at the tumor site, delivering drug-eluting fragments. This study demonstrates the effect of shell characteristics and MB functionalization (TRAIL-ligated and Dox-loaded MBs) on the acoustic response of MBs, and the cumulative effect of shell type. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3189-3196, 2017.

Many cancer therapy regimes still rely heavily on the systemic administration of toxic chemotherapeutic agents. Ultrasound contrast agents consisting of microbubbles (MBs) have emerged as a drug delivery vehicle to overcome the challenges associated with systemic chemotherapy. Here, we describe the development of non-immunogenic, functionalized polylactic acid (PLA) MBs for use in targeted cancer therapy. Our previous studies have shown that the balance between acoustic behavior and improved immune avoidance was scalable and successful to different degrees with two different PEGylation methods and was best achieved using incorporation of PEG-PLA at 5 wt % and for a LipidPEG at 1 wt %. Capitalizing on this, we now attach a targeting ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which selectively induces tumor cell death upon binding to cancer cell-specific surface receptors, initiating a transmembrane apoptosis signal. Additionally, the functionalized MBs were designed to coencapsulate doxorubicin (Dox) that can be released from the polymer shell in response to ultrasound focused at the tumor site, shielding healthy tissues from toxicity while increasing the potency and efficiency of treatment to the tumor tissue. Ligation of TRAIL reduced the encapsulation efficiency for Dox compared to those of their non-ligated counterparts (p0.0001) by approximately 34% for 100% PLA, 23% for 5 wt % PEG-PLA, and 30% for the 1 wt % LipidPEG platform. All platforms exhibited a burst effect (7%, p0.0001), and sustained release lasted for over 150 h. This work has resulted in a choice of effective ultrasound-triggered, non-immunogenic, targeted drug delivery agents for potential use in cancer therapy. These platforms have many advantages over the systemic administration of chemotherapeutic drugs and represent a promising treatment to better serve the population with solid cancerous tumors as a whole.

OBJECTIVES—: Hypoxic cancer cells have been shown to be more resistant to radiation therapy than normoxic cells. Hence, this study investigated whether ultrasound (US)-induced rupture of oxygen-carrying microbubbles (MBs) would enhance the response of breast cancer metastases to radiation. METHODS—: Nude mice (n = 15) received stereotactic injections of brain-seeking MDA-MB-231 breast cancer cells into the right hemisphere. Animals were randomly assigned into 1 of 5 treatment groups: no intervention, 10 Gy radiation using a small-animal radiation research platform, nitrogen-carrying MBs combined with US-mediated MB rupture immediately before 10 Gy radiation, oxygen-carrying MBs immediately before 10 Gy radiation, and oxygen-carrying MBs with US-mediated MB rupture immediately before 10 Gy radiation. Tumor progression was monitored with 3-dimensional US, and overall survival was noted. RESULTS—: All groups except those treated with oxygen-carrying MB rupture and radiation had continued rapid tumor growth after treatment. Tumors treated with radiation alone showed a mean increase in volume ± SD of 337% ± 214% during the week after treatment. Tumors treated with oxygen-carrying MBs and radiation without MB rupture showed an increase in volume of 383% ± 226%. Tumors treated with radiation immediately after rupture of oxygen-carrying MBs showed an increase in volume of only 41% ± 1% (P = 0.045), and this group also showed a 1 week increase in survival time. CONCLUSIONS—: Adding US-ruptured oxygen-carrying MBs to radiation therapy appears to delay tumor progression and improve survival in a murine model of metastatic breast cancer.

Ultrasound contrast agents are typically microbubbles (MB) with a gas core that is stabilized by a shell made of lipids, proteins, or polymers. The high impedance mismatch between the gas core and an aqueous environment produces strong contrast in ultrasound (US). Poly(lactic acid) (PLA) MB, previously developed in our laboratory, have been shown to be highly echogenic both in vitro and in vivo. Combining US with other imaging modalities such as fluorescence, magnetic resonance imaging (MRI), or computerized tomography (CT) could improve the accuracy of many US applications and provide more comprehensive diagnostic information. Furthermore, our MB have the capacity to house a drug in the PLA shell and create drug-loaded nanoparticles in situ when passing through an ultrasound beam. To create multimodal contrast agents, we hypothesized that the polymer shell of our PLA MB platform could accommodate additional payloads. In this study, we therefore modified our current MB by encapsulating nanoparticles including aqueous or organic quantum dots (QD), magnetic iron oxide nanoparticles (MNP), or gold nanoparticles (AuNP) to create bimodality platforms in a manner that minimally compromised the performance of each individual imaging technique.

To improve visualization of malignant regions in the colon epithelium during diagnostic procedures, we have recently suggested a multimodal system comprising the water-soluble cationized polyacrylamide, tagged with the near infrared dye derivative IR-783-S-Ph-COOH [fluorescent-cationized polyacrylamide (Flu-CPAA)] and conjugated to the recognition peptide VRPMPLQ to form Flu-CPAA-Pep. The fluorescent-cationized polyacrylamide-peptide conjugate (Flu-CPAA-Pep) is then incorporated into echogenic microbubbles (MBs) made of polylactic acid (PLA) to protect it from pre-mature interactions with plasma proteins upon intravenous administration. It is expected that under directed ultrasound interrogation the Flu-CPAA-Pep cargo would be released in the region of interest, as a result of the MBs rupture into submicron PLA fragments (SPF). Due to their nanoscale dimension, the SPF will escape from the vasculature and allow a specific binding of the Flu-CPAA-Pep to the suspected malignant tissue. The complex nature of the system requires a multifaceted statistically based experimental design. Here we apply a design of experiment methodology to enable effective screening of key parameters in our experimental setup. It enables the assessment of the role of the different formulation parameters by identifying statistically significant interactions as observed in vitro (cell lines) and in vivo (colon cancer-induced rat model). Particularly important was the identification of the interaction between the fraction (mol%) of the cationic monomer in the Flu-CPAA and (a) the presence of recognition peptide as assessed in the in vitro experiments and (b) the use of a presenting platform (whether MBs or SPF) as assessed in the in vivo experiments. Copyright © 2015 John Wiley & Sons, Ltd.

To increase colonoscopy capability to discriminate benign from malignant polyps, we suggest combining two imaging approaches based on targeted polymeric platforms. Water-soluble cationized polyacrylamide (CPAA) was tagged with the near infrared (NIR) dye IR-783-S-Ph-COOH to form Flu–CPAA. The recognition peptide VRPMPLQ (reported to bind specifically to CRC tissues) was then conjugated with the Flu–CPAA to form Flu–CPAA–Pep which was then incorporated into echogenic microbubbles (MBs) made of polylactic acid (PLA) that are highly responsive to ultrasound. The ultimate design includes intravenous administration combined with local ultrasound and intra-colon inspection at the NIR range. In this proof of principle study PLA MBs were prepared by the double emulsion technique and loaded with several types of Flu–CPAA–Pep polymers. After insonation the submicron PLA fragments (SPF)-containing Flu–CPAA–Pep were examined in vitro for their ability to attach to colon cancer cells and in vivo (DMH induced rat model) for their ability to attach to colon malignant tissues and compared to the specific attachment of the free Flu–CPAA–Pep. The generation of SPF-containing Flu–CPAA–Pep resulted in a tissue attachment similar to that of the free, unloaded Flu–CPAA–Pep. The addition of VRPMPLQ to the polymeric backbone of the Flu–CPAA reduced cytotoxicity and improved the specific binding.

Radiation therapy is frequently used in the treatment of malignancies, but tumors are often more resistant than the surrounding normal tissue to radiation effects, because the tumor microenvironment is hypoxic. This manuscript details the fabrication and characterization of an ultrasound-sensitive, injectable oxygen microbubble platform (SE61(O2)) for overcoming tumor hypoxia. SE61(O2) was fabricated by first sonicating a mixture of Span 60 and water-soluble vitamin E purged with perfluorocarbon gas. SE61(O2) microbubbles were separated from the foam by flotation, then freeze dried under vacuum to remove all perfluorocarbon, and reconstituted with oxygen. Visually, SE61(O2) microbubbles were smooth, spherical, with an average diameter of 3.1 μm and were reconstituted to a concentration of 6.5 E7 microbubbles/ml. Oxygen-filled SE61(O2) provides 16.9 ± 1.0 dB of enhancement at a dose of 880 μl/l (5.7 E7 microbubbles/l) with a half-life under insonation of approximately 15min. In in vitro release experiments, 2 ml of SE61(O2) (1.3 E8 microbubbles) triggered with ultrasound was found to elevate oxygen partial pressures of 100 ml of degassed saline 13.8 mmHg more than untriggered bubbles and 20.6 mmHg more than ultrasound triggered nitrogen-filled bubbles. In preliminary in vivo delivery experiments, triggered SE61(O2) resulted in a 30.4 mmHg and 27.4 mmHg increase in oxygen partial pressures in two breast tumor mouse xenografts.

This study represents the first attempt to combine surface TRAIL expression and doxorubicin co-encapsulation in a single drug delivery agent in the form of ultrasound-responsive microbubbles that shatter into fragments, or nanoshards, in an ultrasound beam. We compare customized microbubbles of different polymeric shell compositions, and investigate the effect of both shell composition and incorporation of doxorubicin on action against TRAIL-sensitive MDA-MB-231 and TRAIL-resistant MCF7 human breast adenocarcinoma cells. Ligation of TRAIL only significantly impacted MDA-MB-231 cells predominantly by apoptosis, and had minimal effect on MCF12A (normal control) cells. For all shell types, nanoshards had a greater effect (apoptotic death ranging from approximately 25% for 1 wt % LipidPEG to 50% for 100% PLA), reflecting the greater surface area and larger number of particles that ultrasound generates. Encapsulation of doxorubicin generated necrosis in all cell lines, but PEGylation produced less effective necrosis in all cell lines. Co-encapsulation of doxorubicin within the contrast agent shell increased MDA-MB-231 cell death to approximately 40-80%, representing a marked increase over TRAIL alone, reflecting the dramatic effect of shell composition. Additionally, shells that co-encapsulated TRAIL and doxorubicin resulted in approximately 30-60% death in TRAIL-resistant MCF7 human breast adenocarcinoma cells, compared with little apoptotic response in these cells from shells encapsulating TRAIL alone, demonstrating the sensitization effect of the drug. This work has resulted in production of a library of effective ultrasound-triggered, minimally immunogenic, targeted drug delivery agents for potential use in cancer therapy, and represents a promising multifaceted treatment to better serve the population with solid tumors. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1903-1915, 2018.

Studies have shown that treatment of pancreatic ductal adenocarcinoma (PDAC) with systemic administration of gemcitabine (GEM) is inhibited by excessive desmoplastic connective tissue surrounding the tumor. Ultrasound (US) insonification combined with drug-loaded poly lactic acid (PLA) microbubbles (MB) may circumvent this through targeted delivery to the tumor via increased permeability and lodging of GEM-loaded MB fragments in the tumor tissue. GEM-loaded MB were fabricated through a double emulsion process, and evaluated for efficacy as potential drug delivery agents. Acoustic and physical characterization suggest that GEM encapsulation does not significantly affect MB morphology or contrast activity (p > 0.23 compared with unmodified MB). Inertial cavitation upon exposure to US is not impacted by drug encapsulation, indicated by GEM-loaded MB exhibiting significant size reduction to fragments approximately 400 nm in diameter following insonation (p 0.1). Encapsulation of GEM within the PLA shell of MB resulted in viable drug delivery agents for PDAC treatment.

Tumor hypoxia has been shown to decrease the sensitivity of solid tumors to radiation. Systemic efforts to increase tumor oxygenation levels immediately prior to therapy, however, have largely proven unsuccessful. The objective of this work was to determine the utility of oxygen-filled, ultrasound sensitive microbubbles for locally delivering oxygen and sensitizing tissue to radiation in a breast cancer model.

In the past decade, two formerly estranged domains, spirituality and work, have been increasingly linked. Why has this happened? This articles posits that this strange union is an unavoidable consequence of living in a turbulent time. As our world grows more chaotic and unpredictable, leaders must respond to questions that have historically only been answered through spiritual traditions: How do I cope with incertainty? How do I help others find meaning in their lives? What are my values? How can I act with courage and integrity? Leaders also play a key role in helping people recognize that life is cyclical, unpredictable, and unstable and that they must engage it as such. This is well-explored in spiritual traditions, but not in traditional management literature. Several organizational and personal practices are offered as simple ways to develop leadership capacity for leading in turbulence.

The composition of microcapsules designed for drug delivery significantly impacts their properties. Ultrasound contrast agents, consisting of stabilized microbubbles (MBs), have emerged as versatile potential drug delivery vehicles to both image and overcome challenges associated with systemic chemotherapy. In our development of polylactic acid MBs decorated with immune-shielding polyethylene glycol chains, we have shown that the balance between acoustic behavior and immune avoidance was scalable and amenable to two distinct PEGylation methods, either incorporation of 5 wt% PEGylated PLA or insertion of 1 wt% PEGylated lipid (LipidPEG) in the polymeric shell. Here we describe the effects of shell compositions on MB functionalization for use in targeted cancer therapy. We chose tumor necrosis factor-related apoptosis inducing ligand (TRAIL) as the targeting ligand, motivated by the ability to both target cells and selectively induce tumor cell death upon binding. Additionally, the MBs were designed to co-encapsulate the chemotherapeutic doxorubicin (Dox) within the shell that works with TRAIL to sensitize resistant cells. We have previously shown that the MBs shatter in response to ultrasound focused at the tumor site, delivering drug-eluting fragments. This study demonstrates the effect of shell characteristics and MB functionalization (TRAIL-ligated and Dox-loaded MBs) on the acoustic response of MBs, and the cumulative effect of shell type. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3189-3196, 2017.

The stabilizing encapsulation of a microbubble-based ultrasound contrast agent (UCA) critically affects its acoustic properties. Polymers, which behave differently from materials commonly used ( i.e., lipids or proteins) for monolayer encapsulation, have the potential for better stability and improved control of encapsulation properties. Air-filled microbubbles coated with poly(DL-lactic acid) (PLA) are characterized here using in vitro acoustic experiments and several models of encapsulation. The interfacial rheological properties of the encapsulation are determined according to each model using attenuation of ultrasound through a suspension of microbubbles. Then the model predictions are compared with scattered non-linear (sub- and second harmonic) responses. For this microbubble population (average diameter, 1.9 μm), the peak in attenuation measurement indicates a weighted-average resonance frequency of 2.5–3 MHz, which, in contrast to other encapsulated microbubbles, is lower than the resonance frequency of a free bubble of similar size (diameter, 1.9 μm). This apparently contradictory result stems from the extremely low surface dilational elasticity (around 0.01–0.07 N/m) and the reduced surface tension of the poly(DL-lactic acid) encapsulation, as well as the polydispersity of the bubble population. All models considered here are shown to behave similarly even in the non-linear regime because of the low surface dilational elasticity value. Pressure-dependent scattering measurements at two different excitation frequencies (2.25 and 3 MHz) revealed strongly non-linear behavior with 25–30 dB and 5–20 dB enhancements in fundamental and second-harmonic responses, respectively, for a contrast agent concentration of 1.33 μg/mL in the suspension. Sub-harmonic responses are registered above a relatively low generation threshold of 100–150 kPa, with up to 20 dB enhancement beyond that pressure. Numerical predictions from all models show good agreement with the experimentally measured fundamental response, but not with the experimental second-harmonic response. The characteristic features of sub-harmonic responses and the steady response beyond the threshold are matched well by model predictions. However, prediction of the threshold value depends on estimated properties and size distribution. The variation in size distribution from sample to sample leads to variation in estimates of encapsulation properties: the lowest estimated value for surface dilational viscosity better predicts the sub-harmonic threshold.

In the work described here, gene delivery using polymer microbubbles triggered by ultrasound in vitro was investigated. The effects of pressure amplitude (0–2 MPa), center frequency (1–5 MHz), pulse length (3–12,000 μs), pulse repetition frequency (5–20,000 Hz) and exposure time (0–30 s) on transfection efficiency and cell viability were examined. The effects of radiation force, calcium ion concentration and timing of treatments were also examined. Cells were successfully transfected with pressure amplitudes as low as 250 kPa. Transfection was most efficient at lower frequencies and longer pulse lengths, with a transfection efficiency of 24.2 ± 2.0% achieved using a center frequency of 1 MHz, pressure amplitude of 1 MPa, pulse length of 12,000 μs and pulse repetition frequency of 5 Hz. Gene delivery was also affected by the extracellular calcium ion concentration and the timing of treatments.

Several strategies have been investigated to enhance axonal regeneration after spinal cord injury, however, the resulting growth can be random and disorganized. Bioengineered scaffolds provide a physical substrate for guidance of regenerating axons towards their targets, and can be produced by freeze casting. This technique involves the controlled directional solidification of an aqueous solution or suspension, resulting in a linearly aligned porous structure caused by ice templating. In this study, freeze casting was used to fabricate porous chitosan-alginate (C/A) scaffolds with longitudinally oriented channels. Chick dorsal root ganglia explants adhered to and extended neurites through the scaffold in parallel alignment with the channel direction. Surface adsorption of a polycation and laminin promoted significantly longer neurite growth than the uncoated scaffold (poly-L-ornithine + Laminin = 793.2 ± 187.2 μm; poly-L-lysine + Laminin = 768.7 ± 241.2 μm; uncoated scaffold = 22.52 ± 50.14 μm) (P < 0.001). The elastic modulus of the hydrated scaffold was determined to be 5.08 ± 0.61 kPa, comparable to reported spinal cord values. The present data suggested that this C/A scaffold is a promising candidate for use as a nerve guidance scaffold, because of its ability to support neuronal attachment and the linearly aligned growth of DRG neurites.

Wheatley contends that as a global culture, we are essentially lost in an unsatisfying world of “hyper speed, hyper stress, and hyper irrationality.” Yet many may not recognize being lost, because they first deny it is happening. She suggests that leaders must create new maps, which first entails admitting that we are lost, in order to create something new and better. This leads to an ongoing process of experimentation, reflecting and learning, and taking in new information and diverse perspectives. The process “needs to be a collective recognition, not just an announcement by the leader.”

Microbubbles, developed as ultrasound contrast agents for vascular imaging are valuable theranostic agents for multiple biomedical applications. The utility of these agents can be attributed to their unique response to ultrasound. When exposed to an acoustic pulse, microbubbles can resonate to create a strong acoustic signal that can be used to diagnose conditions including cardiac abnormalities and cancerous lesions. Microbubble resonance can also generate powerful and localized forces capable of creating pores in cell membranes and blood vessels that can be used for targeted drug and gene delivery as well as thromoblysis and opening of the blood brain barrier. Microbubbles have been encapsulated with a variety of materials including lipids, proteins and polymers, these materials have been shown to control a microbubble's acoustic properties as well as its effectiveness as a drug carrier. This review focuses on the progress of microbubbles and the applications of microbubbles as theragnostic agents.

In order for site-directed polymer ultrasound contrast agents (UCAs) to provide acoustic enhancement at disease sites to distinguish normal tissue from diseased tissue, the surface of these agents must be functionalized with mixtures of grafted polymers. Here a combination of longer liganded polyethylene glycol (PEG)-lipids and shorter unliganded PEG-lipids were introduced into the oil phase of a modified solvent evaporation double emulsion method for preparing UCAs. UCAs with different lengths of both liganded and unliganded lipids were imaged under 7.5MHz ultrasound. The B-mode image brightness of the mixed PEG-lipid UCAs was within 1dB the brightness of the unliganded surface. After 15min of continuous insonation, 70% of the contrast signal remained. The peptide arginine- glycine-aspartic acid(RGD) wasadded to the surface ofthese UCAs through a biotin-avidin linkage and binding was assessed under static and shear conditions. Binding was significant after 30min of static incubation and the adher- ence of the UCA increased under shearflow from 3 UCA/cell (static) to 5 UCA/cell (shear). Biotechnol. Bioeng. 2010;106: 501-506.

Successful spinal cord repair is thought to be promoted with hierarchically structured scaffolds. These should combine aligned porosity with additional linear features on the micrometer scale to guide axons across multiple length scales. Such scaffolds are generated through the carefully controlled directional solidification of an aqueous biopolymer solution, followed by lyophilization. Under specific freezing conditions this yields a highly regular and aligned lamellar architecture. This architecture exhibits uniform ridges of controlled height and width on the lamellar surface. These ridges run parallel to the pore axis, serving as secondary guidance features. The ridges are capable of linearly aligning 62.4% of chick dorsal root ganglia neurites to within ±10° of the ridge direction. Notably, neurites sprouting perpendicular to the ridge are guided into alignment with these microridged features.

All of us love the idea of a hero, someone who will do the heavy lifting and magically fix our problems. But waiting for a savior is counterproductive, the authors contend. Instead they introduce the model of “leaders-as-hosts.” This more inclusive leader is more suited to grappling with the big, seemingly insoluble problems routinely faced by organizations, communities, and countries. They point to examples of the successful implementation of this model in Columbus, Ohio, in dealing with such issues as healthcare, homelessness, and hunger.

A polymer ultrasound contrast agent (UCA) developed in our lab has been shown to greatly reduce in size when exposed to ultrasound, resulting in nanoparticles less than 400 nm in diameter capable of escaping the leaky vasculature of a tumor to provide a sustained release of drug. Previous studies with the hydrophilic drug doxorubicin (DOX) demonstrated enhanced drug delivery to tumors when triggered with ultrasound. However the therapeutic potential has been limited due to the relatively low payload of DOX. This study compares the effects of loading the hydrophobic drug paclitaxel (PTX) on the agent's acoustic properties, drug payload, tumoricidal activity, and the ability to deliver drugs through 400 nm pores. A maximum payload of 129.46 ± 1.80 μg PTX/mg UCA (encapsulation efficiency 71.92 ± 0.99%) was achieved, 20 times greater than the maximum payload of DOX (6.2 μg/mg), while maintaining the acoustic properties. In vitro, the tumoricidal activity of paclitaxel loaded UCA exposed to ultrasound was significantly greater than controls not exposed to ultrasound (p < 0.0016). This study has shown that PTX loaded UCA triggered with focused ultrasound have the potential to provide a targeted and sustained delivery of drug to tumors.

Current influenza virus vaccines primarily elicit antibodies and can be rendered ineffective by antigenic drift and shift. Vaccines that elicit CD8+ T cell responses targeting less variable proteins may function as universal vaccines that have broad reactivity against different influenza virus strains. To generate such a universal vaccine, we encapsulated live influenza virus in a biopolymer and delivered it to mice subcutaneously. This vaccine was safe, induced potent CD8+ T cell immunity and protected mice against heterosubtypic lethal challenge. Safety of subcutaneous (SQ) vaccination was tested in Rag2−/−γc−/− double knockout mice which we show cannot control intranasal infection. Biopolymer encapsulation of live influenza virus could be used to develop universal CD8+ T cell vaccines against heterosubtypic and pandemic strains.

Engineered tissue strategies for central nervous system (CNS) repair have the potential for localizing treatment using a wide variety of cells or growth factors. However, these strategies are often limited by their ability to address only one aspect of the injury. Here we report the development of a novel alginate construct that acts as a multi-functional tissue scaffold for CNS repair, and as a localized growth factor delivery vehicle. We show that the surface of this alginate construct acts as an optimal growth environment for neural progenitor cell (NPC) attachment, survival, migration, and differentiation. Importantly, we show that tailor-made alginate constructs containing brain-derived neurotrophic factor or neurotrophin-3 differentially direct lineage fates of NPCs and may therefore be useful in treating a wide variety of injuries. It is this potential for directed differentiation of a scaffold prior to implantation at the injury site that we explore here.

The perfluorcarbon (perfluorobutane) ultrasound contrast agent ST68, composed of sonicated mixtures of non-ionic surfactants, is stable in solution for only a few weeks at 4°C. Freeze-drying critically diminished ST68’s ability to reflect ultrasound (its echogenicity). A method of incorporating specific lyoprotectants before lyophilization was investigated. Reintroduction of perfluorobutane to the protected freeze-dried sample, followed by reconstituting with preserved echogenicity. Glucose, trehalose, sucrose, and mannitol were tested at 100 mM and in vitro echogenicity data was collected from samples with dose concentrations of 50 µl/l to 300 µl/l. Glucose was found to be the best lyoprotectant providing an average (n=3) maximum peak enhancement of 23.2 ± 1.2 dB in vitro, measured at 5 MHz, 684 kPa, and a pulse repetition frequency (PRF) of 100 Hz (p

Low delivery efficiency combined with systemic toxicity of traditional chemotherapy provides a need for improved chemotherapeutic delivery. Within our laboratory, we have developed polymer ultrasound contrast agents (1.2-1.8 mum in diameter) containing doxorubicin (Dox) within the shell (100-150 nm). In vivo this platform is expected to circulate through the vasculature until activated at the tumor site with external focused ultrasound (US). In vitro, the agent is responsive to US and when insonated at peak positive pressure amplitudes of 0.69 MPa and above, shows dramatic size reduction, eventually reaching a mean particle size of 350 nm, presumably due to fragmentation of, or gas release from the agent. The resulting Dox-polymer particles retain the drug and are small enough to pass through the leaky pores (350-400 nm) within the tumor vasculature, providing a sustained intratumoral release of chemotherapeutic as the polymer degrades. In vivo studies using a VX2 liver tumor model have shown that the combination of the agent and US results in nearly 50% less drug delivered to the nontargeted, healthy liver ( p = 0.009) and a 110% increase ( p = 0.004) in Dox delivery to the viable peripheral tissue of the tumor, relative to the uninsonated controls. This study shows how US-mediated destruction of drug-loaded polymer contrast agent can be used to deliver encapsulated drug for potential sustained release. Penetration mechanisms of these resulting particles and their ability to provide a sustained release from the tumor interstia will be explored in the future.

Gas-filled microbubbles are used as contrast agents in diagnostic ultrasound imaging. A preclinical, acute toxicity study of 2 surfactant-stabilized ultrasound contrast agents (ST68 and ST44) was conducted. Subjects were 104 Sprague-Dawley rats (experimental doses, 0.1, 0.2, 0.8, and 1.0 mL/kg; control, 1.0 mL/kg saline) that were studied for 14 days after contrast; clinical signs, weight, blood, and urine were evaluated. Histopathology was performed following euthanasia. Of the 40 animals receiving ST44, 4 died prematurely and a dose dependency was demonstrated ( P = .011), whereas in the ST68 groups only 1 death occurred (no dose dependency; P = .48). Only the weight of rats injected with ST44 varied significantly ( P = .0003). This dependency was also found for 3 of 5 urine parameters and 4 of 36 blood parameters ( P < .05). For ST68, only 1 urine parameter showed significance ( P < .0001). Giant cell infiltration in the lungs was significantly higher than controls in the ST44 0.1 mL/kg and the ST68 0.8-1.0 mL/kg groups ( P < .01). It is concluded that the prudent choice for future nonrodent, toxicology studies and potentially for human clinical trials is ST68 (given the deaths in the ST44 groups).

Poly lactic acid (PLA) ultrasound contrast agents (CA) have been developed previously in our laboratory for ultrasound (US) imaging, as well as surface coated with doxorubicin to create a potential targeted platform of chemotherapeutic delivery using focused US. However, we have previously found it impossible to sterilize these agents while at the same time maintaining their acoustic properties, a task that would probably require fabrication within a clean facility. The purpose of this paper is to investigate the feasibility of using plasma to sterilize these CA while maintaining maximum echogenicity, a step that would greatly facilitate in vivo investigations. Effects of plasma exposure time (1, 3 and 6 min) and intensity (low—10 mA, 6.8 W; medium—15 mA, 10.5 W; and high—25 mA, 18 W) on the CAs' acoustic properties, surface morphology, zeta potential, capacity to carry chemotherapeutics and overall sterility are described. Both increases in plasma intensity and exposure time increased CA zeta potential and also significantly increased drug payload. High-intensity plasma exposure for 3 min was found to be an optimal sterilization protocol for maximal (100%) preservation of CA echogenicity. Plasma exposure resulted in sterile samples and maintained original CA enhancement of 20 dB and acoustic half-life over 75 min, while increasing CA zeta potential by 11 mV and doxorubicin loading efficiency by 10%. This study not only shows how a highly temperature- and pressure-sensitive agent can be sterilized using plasma, but also that surface modification can be used to increase surface binding of the drug. (E-mail: wheatley@coe.drexel.edu )

People often comment that the new leadership models derived from living systems and complexity science couldn’t possibly work in “the real world.” I assume they are referring to their organization or government, which they experience as a predesigned bureaucracy, governed by policies and laws, where people are expected to do what they’re told and wait for instructions. This “real world” of mechanistic organizations craves efficiency and obedience. It relies on standard operating procedures for every situation, even when chaos erupts and things are out of control. This is not the real world. This world is a man-made, dangerous fiction that destroys our capacity to deal well with what’s really going on. The real real world, not this fake one, demands that we learn to cope with chaos, that we know how to evoke human ingenuity and skills, that we adopt strategies and behaviors that lead to order, not to more chaos. In this historic moment, we live caught between a worldview that no longer works and a new one that seems too bizarre to contemplate. To expose this, I want to apply the lens of new science to one of free society’s most compelling, real world challenges: How well we understand and respond to global terror networks. Using this new paradigm, high resolution lens, many dynamics become visible that are crucial to understand, yet were obscured from view by our old sight.

Ultrasound contrast agents (CA) consist of stabilized gas bubbles that, when injected intravenously, provide an acoustic impedance mismatch, producing additional contrast to a diagnostic ultrasound scan. These agents must be smaller than 8 μm in order to pass safely through the capillaries, contain gas for an impedance mismatch and should be stable enough to survive the duration of the imaging session. A double emulsion technique has previously been optimized within our laboratory to create CA with 50:50 poly (lactic-co-glycolic acid) (PLGA). Although a great deal of research has focused on the effects of molecular weight and end capping on solid PLGA particles, very little has been done to examine the effects of these parameters on hollow CAs formed in a double emulsion. Non-end capped PLGA was found to provide maximum enhancement at a molecular weight of 66.0 kDa, giving an ultrasound enhancement of roughly 18.5 dB. The enhancement demonstrated by CA formed using the end-capped PLGA rose to a maximum enhancement of 19 dB at the highest commercially available molecular weight of 82.4 kDa. A strong correlation was seen between ultrasound enhancement, stability under ultrasonic conditions, surface morphology and zeta potential. This study shows the influence of polymer characteristics on the resulting properties of CA and the ability to tailor CAs to particular applications by varying the polymer choice. POLYM. ENG. SCI., 2008. © 2008 Society of Plastics Engineers

Ultrasound Contrast Agents (UCA) are microencapsulated gas bubbles used to increase the overall contrast of the ultrasound (US) image. Novel multimodal polymeric UCA containing additional contrast agents that are active in multiple imaging modalities have been developed. Four platforms of loaded poly (lactic acid) (PLA) shelled UCA are presented. The first two platforms co-encapsulate aqueous or organic Quantum Dots (QD). The third platform contains magnetic iron oxide nanoparticles (MNP). The fourth platform contains gold nanoparticles (AuNP). All platforms showed that the properties of UCA are maintained as well as the properties of additional encapsulated contrast agent. This study explores the potential application for improving the screening of a subject using at least two different modalities.

This research aims to develop an injectable polymer-based platform to enable minimally-invasive targeted delivery of drug-loaded nano particles. Studies have shown that polymer-stabilized gas microbubbles are effective in enhancing an ultrasound image, especially those involving cancerous tumors. These contrast agents can serve a dual purpose when designed to encapsulate a drug within the shell, providing both diagnostic and therapeutic applications. The current gold standard treatment of non-resectable pancreatic ductal adenocarcinoma (PDA) is gemcitabine (GEM), which is administered systemically. However, response rates are low because the tumor environment inhibits the drug, especially the stroma surrounding the tumor. We hypothesize that GEM-loaded microbubbles injected intravenously will pass through the stroma and, when exposed to ultrasound (US), will burst to form nanoshards (n-Sh) which will lodge within the pancreatic tumor tissue, undergo sustained release of encapsulated GEM, and lead to cancer cell death through targeted therapy.

Many injectables are not amenable to standard sterilization methods, which destroy sensitive materials. This is particularly true for ultrasound contrast agents (UCA) consisting of gas bubbles stabilized by a surfactant or polymer shell. We investigated a new method to achieve safe and effective sterilization in production by introducing dielectric-barrier discharge non-thermal plasma. A dielectric-barrier discharge was generated to first produce plasma-treated phosphate-buffered saline (PTPBS), which was used as a sterilant solution for our UCA SE61, avoiding direct heat, pressure, chemicals, or radiation. Treated samples were tested for acoustic properties in vitro and in a flow phantom, and for sterility by standard methods. Three minutes plasma treatment of phosphate-buffered saline (PBS) proved effective. The samples showed significant inactivation of inoculated bacteria upon PTPBS treatment as compared to un-treated-PBS (p = 0.0022). The treated and untreated samples showed no statistical significance (p > 0.05) in acoustic response or bubble diameter (mean ± SEM: 2.52 ± 0.31 μm). Nile Red was used to model intercalation of drug in the hydrophobic shell, intercalated successfully into SE61, and was unaffected by plasma treatment. The PTPBS completely sterilized suspensions of UCA, and it did not compromise the acoustic properties of the agent or its ability to retain a hydrophobic compound.

The increasing risk of incidental exposure to nanomaterials has led to mounting concerns regarding nanotoxicity. Zinc oxide nanoparticles (ZnO NPs) are produced in large quantities and have come under scrutiny due to their capacity to cause cytotoxicity in vitro and potential to cause harm in vivo. Recent evidence has indicated that ZnO NPs promote autophagy in cells; however, the signaling pathways and the role of ion release inducing toxicity remain unclear. In this study, we report that ZnO NPs are immunotoxic to primary and immortalized immune cells. Importantly, such immunotoxicity is observed in mice in vivo, since death of splenocytes is seen after intranasal exposure to ZnO NPs. We determined that ZnO NPs release free Zn(2+) that can be taken up by immune cells, resulting in cell death. Inhibiting free Zn(2+) ions in solution with EDTA or their uptake with CaCl2 abrogates ZnO NP-induced cell death. ZnO NP-mediated immune cell death was associated with increased levels of intracellular reactive oxygen species (ROS). ZnO NP death was not due to apoptosis, necroptosis or pyroptosis. Exposure of immune cells to ZnO NPs resulted in autophagic death and increased levels of LC3A, an essential component of autophagic vacuoles. Accordingly, ZnO NP-mediated upregulation of LC3A and induction of immune cell death were inhibited by blocking autophagy and ROS production. We conclude that release of Zn(2+) from ZnO NPs triggers the production of excessive intracellular ROS, resulting in autophagic death of immune cells. Our findings suggest that exposure to ZnO NPs has the potential to impact host immunity.