Influence of Phase Change Droplet Activation and Microbubble Cavitation on the Microenvironment of Hepatocellular Carcinoma.

OBJECTIVE: Both microbubble ultrasound contrast agents and acoustic phase change droplets (APCD) have been explored in hepatocellular carcinoma (HCC). This work aimed to evaluate changes to the HCC microenvironment following either microbubble or APCD destruction in a syngeneic pre-clinical model. METHODS: Mouse RIL-175 HCC tumors were grown in the right flank of 64 immunocompetent mice. Pre-treatment, photoacoustic volumetric tumor oxygenation, and power Doppler measurements were obtained using a Vevo 3100 system (VisualSonics, Toronto, Canada). The experimental groups received a 0.1 mL bolus injection of either Definity ultrasound contrast agent (Lantheus Medical Imaging) or APCD fabricated by condensing Definity. Following injection, ultrasound destruction was performed using flash-replenishment sequences on a Sequoia with a 10L4 probe (Siemens) for the duration of enhancement. Tumor oxygenation and power Doppler measurements were then repeated immediately post-ultrasound treatment. Twenty-four hours post-treatment, animals were euthanized, and tumors were harvested and stained for CD31, Cleaved Caspase 3 and CD45. RESULTS: Imaging biomarkers demonstrated a significant reduction in percent vascularity following either microbubble or APCD destruction in the tumor microenvironment ( p < 0.022) but no significant changes in tumor oxygenation (p = 0.12). Similarly, immunohistochemistry data demonstrated a significant decrease in CD31 expression (p < 0.042) and an increase in apoptosis (p < 0.014) in tumors treated with destroyed microbubbles or APCD relative to controls. Finally, a significant increase in CD45 expression was observed in tumors treated with APCD (p = 0.046), indicating an increase in tumor immune response. CONCLUSION: Ultrasound-triggered destruction of both microbubbles and APCD reduces vascularity, increases apoptosis, and may also increase immune response in this HCC model.

Determining Ultrasound Parameters for Bursting Polymer Microbubbles for Future Use in Spinal Cord Injury.

OBJECTIVE: We believe our poly(lactic acid) (PLA) microbubbles are well suited for therapeutic delivery to spinal cord injury (SCI) using ultrasound-triggered bursting. We investigated the feasibility of clinical ultrasound bursting in situ, the optimal bursting parameters in vitro and the loading and release of a model bio-active DNA. METHODS: Microbubbles were tested using clinical ultrasound in a rat cadaver SCI model. Burst pressure thresholds were determined using the change in enhancement after ultrasound exposure. Resonance frequency, acoustic enhancement, sizing and morphology were evaluated by comparing two microbubble porogens, ammonium carbonate and ammonium carbamate. Oligonucleotides were loaded into the shell and released using the found optimized ultrasound bursting parameters. RESULTS: In situ imaging and bursting were successful. In vitro bursting thresholds using frequencies 1, 2.25 and 5 MHz were identified between peak negative pressures 0.2 and 0.5 MPa, believed to be safe for spinal cord. The pressure threshold decreased with decreasing frequencies. PLA bursting was optimized near the resonance frequency of 2.5 to 3.0 MHz using 2.25 MHz and not at lower frequencies. PLA microbubbles, initially with a mean size of approximately 2 µm, remained in one piece, collapsed to between 0.5 and 1 µm and did not fragment. Significantly more oligonucleotide was released after ultrasound bursting of loaded microbubbles. Microbubble-sized debris was detected when using ammonium carbamate, leading to inaccurate microbubble concentration measurements. CONCLUSION: PLA microbubbles made with ammonium carbonate and burst at appropriate parameters have the potential to safely improve intrathecal therapeutic delivery to SCI using targeted ultrasound.

Improved Tumor Control Following Radiosensitization with Ultrasound-Sensitive Oxygen Microbubbles and Tumor Mitochondrial Respiration Inhibitors in a Preclinical Model of Head and Neck Cancer.

Tumor hypoxia (oxygen deficiency) is a major contributor to radiotherapy resistance. Ultrasound-sensitive microbubbles containing oxygen have been explored as a mechanism for overcoming tumor hypoxia locally prior to radiotherapy. Previously, our group demonstrated the ability to encapsulate and deliver a pharmacological inhibitor of tumor mitochondrial respiration (lonidamine (LND)), which resulted in ultrasound-sensitive microbubbles loaded with O2 and LND providing prolonged oxygenation relative to oxygenated microbubbles alone. This follow-up study aimed to evaluate the therapeutic response to radiation following the administration of oxygen microbubbles combined with tumor mitochondrial respiration inhibitors in a head and neck squamous cell carcinoma (HNSCC) tumor model. The influences of different radiation dose rates and treatment combinations were also explored. The results demonstrated that the co-delivery of O2 and LND successfully sensitized HNSCC tumors to radiation, and this was also enhanced with oral metformin, significantly slowing tumor growth relative to unsensitized controls (p < 0.01). Microbubble sensitization was also shown to improve overall animal survival. Importantly, effects were found to be radiation dose-rate-dependent, reflecting the transient nature of tumor oxygenation.

Tumoral oxygenation and biodistribution of Lonidamine oxygen microbubbles following localized ultrasound-triggered delivery.

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 (SE61O2) and ultrasound, pO2 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 SE61O2/LND and ultrasound, pO2 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 O2andLNDvia SE61 also showed an improvement of LND biodistribution in both plasma and tumor tissues (p < 0.001). In summary, ultrasound-sensitive microbubbles loaded with O2 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.

Development of a Dual Drug-Loaded, Surfactant-Stabilized Contrast Agent Containing Oxygen.

Co-delivery of cancer therapeutics improves efficacy and encourages synergy, but delivery faces challenges, including multidrug resistance and spatiotemporal distribution of therapeutics. To address these, we added paclitaxel to previously developed acoustically labile, oxygen-core, surfactant-stabilized microbubbles encapsulating lonidamine, with the aim of developing an agent containing both a therapeutic gas and two drugs acting in combination. Upon comparison of unloaded, single-loaded, and dual-loaded microbubbles, size (~1.7 µm) and yield (~2 × 109 microbubbles/mL) (~1.7) were not statistically different, nor were acoustic properties (maximum in vitro enhancements roughly 18 dB, in vitro enhancements roughly 18 dB). Both drugs encapsulated above required doses calculated for head and neck squamous cell carcinoma, the cancer of choice. Interestingly, paclitaxel encapsulation efficiency increased from 1.66% to 3.48% when lonidamine was included. During preparation, the combination of single drug-loaded micelles gave higher encapsulation (µg drug/g microbubbles) than micelles loaded with either drug alone (lonidamine, 104.85 ± 22.87 vs. 87.54 ± 16.41), paclitaxel (187.35 ± 8.38 vs. 136.51 ± 30.66). In vivo intravenous microbubbles produced prompt ultrasound enhancement within tumors lasting 3-5 min, indicating penetration into tumor vasculature. The ability to locally destroy the microbubble within the tumor vasculature was confirmed using a series of higher intensity ultrasound pulses. This ability to locally destroy microbubbles shows therapeutic promise that warrants further investigation.

Shaping the synthesis of surfactant-stabilized oxygen microbubbles to accommodate encapsulated drug.

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.

Gemcitabine-loaded microbubble system for ultrasound imaging and therapy.

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.

Emerging Applications of Ultrasound-Contrast Agents in Radiation Therapy.

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.

Breast Cancer Brain Metastasis Response to Radiation After Microbubble Oxygen Delivery in a Murine Model.

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.

Preserving the Integrity of Surfactant-Stabilized Microbubble Membranes for Localized Oxygen Delivery.

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 (Tg') 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 (Tm) of the TPGS phase from rising above room temperature. The increase in glucose concentration results in a lowering of Tm 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.

Manipulating multifaceted microbubble shell composition to target both TRAIL-sensitive and resistant cells.

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.

Sensitization of Hypoxic Tumors to Radiation Therapy Using Ultrasound-Sensitive Oxygen Microbubbles.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Shell effects on acoustic performance of a drug-delivery system activated by ultrasound.

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.

Drug Delivery from a Multi-faceted Ultrasound Contrast Agent: Influence of Shell Composition.

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 (p < 0.0001) 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%, p < 0.0001), 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.

Strategies for neurotrophin-3 and chondroitinase ABC release from freeze-cast chitosan-alginate nerve-guidance scaffolds.

Freeze casting, or controlled unidirectional solidification, can be used to fabricate chitosan-alginate (C-A) scaffolds with highly aligned porosity that are suitable for use as nerve-guidance channels. To augment the guidance of growth across a spinal cord injury lesion, these scaffolds are now evaluated in vitro to assess their ability to release neurotrophin-3 (NT-3) and chondroitinase ABC (chABC) in a controlled manner. Protein-loaded microcapsules were incorporated into C-A scaffolds prior to freeze casting without affecting the original scaffold architecture. In vitro protein release was not significantly different when comparing protein loaded directly into the scaffolds with release from scaffolds containing incorporated microcapsules. NT-3 was released from the C-A scaffolds for 8 weeks in vitro, while chABC was released for up to 7 weeks. Low total percentages of protein released from the scaffolds over this time period were attributed to limitation of diffusion by the interpenetrating polymer network matrix of the scaffold walls. NT-3 and chABC released from the scaffolds retained bioactivity, as determined by a neurite outgrowth assay, and the promotion of neurite growth across an inhibitory barrier of chondroitin sulphate proteoglycans. This demonstrates the potential of these multifunctional scaffolds for enhancing axonal regeneration through growth-inhibiting glial scars via the sustained release of chABC and NT-3. Copyright © 2014 John Wiley & Sons, Ltd.

Balancing stealth and echogenic properties in an ultrasound contrast agent with drug delivery potential.

Contrast agents are currently being modified to combine diagnostic and therapeutic capabilities. For ultrasound (US) imaging with polymeric contrast agents, it is necessary to modify the shell to create "stealth" microbubbles but without these modifications sacrificing the agent's ability to interact with the focused US beam. We hypothesize that addition of the classic immune shielding molecule polyethylene glycol (PEG) to a polylactide (PLA) microbubble shell will affect the acoustic and physical properties of the resulting agents. In an effort to determine the best formulation to achieve a balance between stealth and acoustic activity, we compared two PEGylation techniques; addition of increasing amounts of PEG-PLA copolymer and employing incorporation of a PEG lipid (LipidPEG) into the shell. Loss of acoustic enhancement occurred in a dose-dependent manner for both types of PEGylated agents (loss of signal occurred at >5 wt% PEG-PLA and >1 wt% LipidPEG), while immune activation was also reduced in a dose-dependent manner for the PEG-PLA agents. This study shows that the balance between acoustic behavior and improved immune avoidance was scalable and successful to different degrees with both PEGylation methods, and was best achieved using for PEG-PLA at 5 wt% and for LipidPEG at 1 wt%. Studies are ongoing to evaluate the best method for the targeting and drug delivery capabilities of these agents for applications in cancer treatment. This study represents the basis for understanding the consequences of making modifications to the native polymeric shell.

Nanoparticle Loaded Polymeric Microbubbles as Contrast Agents for Multimodal Imaging.

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.

Preservation of imaging capability in sensitive ultrasound contrast agents after indirect plasma sterilization.

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.

Acute exposure to ZnO nanoparticles induces autophagic immune cell death.

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.

Development of an ultrasound sensitive oxygen carrier for oxygen delivery to hypoxic tissue.

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 (SE61O2) for overcoming tumor hypoxia. SE61O2 was fabricated by first sonicating a mixture of Span 60 and water-soluble vitamin E purged with perfluorocarbon gas. SE61O2 microbubbles were separated from the foam by flotation, then freeze dried under vacuum to remove all perfluorocarbon, and reconstituted with oxygen. Visually, SE61O2 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 SE61O2 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 15 min. In in vitro release experiments, 2 ml of SE61O2 (1.3 E8 microbubbles) triggered with ultrasound was found to elevate oxygen partial pressures of 100ml 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 SE61O2 resulted in a 30.4 mmHg and 27.4 mmHg increase in oxygen partial pressures in two breast tumor mouse xenografts.