Cryopreserved red blood cells maintain allosteric control of oxygen binding when utilizing trehalose as a cryoprotectant.

One of the most common life-saving medical procedures is a red blood cell (RBC) transfusion. Unfortunately, RBCs for transfusion have a limited shelf life after donation due to detrimental storage effects on their morphological and biochemical properties. Inspired by nature, a biomimetics approach was developed to preserve RBCs for long-term storage using compounds found in animals with a natural propensity to survive in a frozen or desiccated state for decades. Trehalose was employed as a cryoprotective agent and added to the extracellular freezing solution of porcine RBCs. Slow cooling (-1 °C min-1) resulted in almost complete hemolysis (1 ± 1 % RBC recovery), and rapid cooling rates had to be used to achieve satisfactory cryopreservation outcomes. After rapid cooling, the highest percentage of RBC recovery was obtained by plunging in liquid nitrogen and thawing at 55 °C, using a cryopreservation solution containing 300 mM trehalose. Under these conditions, 88 ± 8 % of processed RBCs were recovered and retained hemoglobin (14 ± 2 % hemolysis). Hemoglobin's oxygen-binding properties of cryopreserved RBCs were not significantly different to unfrozen controls and was allosterically regulated by 2,3-bisphosphoglycerate. These data indicate the feasibility of using trehalose instead of glycerol as a cryoprotective compound for RBCs. In contrast to glycerol, trehalose-preserved RBCs can potentially be transfused without time-consuming washing steps, which significantly facilitates the usage of cryopreserved transfusible units in trauma situations when time is of the essence.

Comparison of Acoustofluidic and Static Systems for Ultrasound-Mediated Molecular Delivery to T Lymphocytes.

Continuous-flow acoustofluidic technologies can potentially improve processing of T lymphocytes for cell therapies by addressing the limitations with viral and non-viral delivery methods. The objective of this study was to assess the intracellular delivery efficiency with acoustofluidic treatment compared with that of static ultrasound treatment. Optimization of parameters in acoustofluidic and static configurations was performed by assessing intracellular delivery of a fluorescent compound (calcein) in viable human Jurkat T lymphocytes. Ultrasound pressure and the concentration of cationic phospholipid-coated microbubbles influenced calcein delivery in both systems. In the static system, a treatment time of 45 s increased molecular delivery compared with 0-30 s (p < 0.01). Refined parameters were used to assess molecular delivery of small and large compounds (0.6-kDa calcein and 150-kDa fluorescein isothiocyanate-dextran, respectively) after ultrasound treatment with the acoustofluidic or static systems. Molecular delivery was similar with refined parameters for acoustofluidic treatment and static treatment (p > 0.05), even though acoustofluidic treatment had lower microbubble concentration (24 µg/mL vs. 94 µg/mL) and shorter treatment time (∼2-3 s vs. 45 s). This study indicates that the acoustofluidic system can significantly enhance intracellular molecular delivery, which could potentially enable acoustofluidic cell transfection during continuous flow processing for manufacture of cell therapies or other applications.

Development of Inspired Therapeutics Pediatric VAD: Computational Analysis and Characterization of VAD V3.

PURPOSE: Pediatric heart failure patients remain in critical need of a dedicated mechanical circulatory support (MCS) solution as development efforts for specific pediatric devices continue to fall behind those for the adult population. The Inspired Pediatric VAD is being developed as a pediatric specific MCS solution to provide up to 30-days of circulatory or respiratory support in a compact modular package that could allow for patient ambulation during treatment. METHODS: Hydrodynamic performance (flows, pressures), impeller/rotor mechanical properties (torques, forces), and flow shear stress and residence time distributions of the latest design version, Inspired Pediatric VAD V3, were numerically predicted and investigated using computational fluid dynamics (CFD) software (SolidWorks Flow Simulator). RESULTS: Hydrodynamic performance was numerically predicted, indicating no change in flow and pressure head compared to the previous device design (V2), while displaying increased impeller/rotor torques and translation forces enabled by improved geometry. Shear stress and flow residence time volumetric distributions are presented over a range of pump rotational speeds and flow rates. At the lowest pump operating point (3000 RPM, 0.50 L/min, 75 mmHg), 79% of the pump volume was in the shear stress range of 0-10 Pa with < 1% of the volume in the critical range of 150-1000 Pa for blood damage. At higher speed and flow (5000 RPM, 3.50 L/min, 176 mmHg), 65% of the volume resided in the 0-10 Pa range compared to 2.3% at 150-1000 Pa. CONCLUSIONS: The initial computational characterization of the Inspired Pediatric VAD V3 is encouraging and future work will include device prototype testing in a mock circulatory loop and acute large animal model.

Development of Inspired Therapeutics Pediatric VAD: Benchtop Evaluation of Impeller Performance and Torques for MagLev Motor Design.

PURPOSE: Despite the availability of first-generation extracorporeal mechanical circulatory support (MCS) systems that are widely used throughout the world, there is a need for the next generation of smaller, more portable devices (designed without cables and a minimal number of connectors) that can be used in all in-hospital and transport settings to support patients in heart failure. Moreover, a system that can be universally used for all indications for use including cardiopulmonary bypass (CPB), uni- or biventricular support (VAD), extracorporeal membrane oxygenation (ECMO) and respiratory assist that is suitable for use for adult, neonate, and pediatric patients is desirable. Providing a single, well designed, universal technology could reduce the incidence of human errors by limiting the need for training of hospital staff on a single system for a variety of indications throughout the hospital rather than having to train on multiple complex systems. The objective of this manuscript is to describe preliminary research to develop the first prototype pump for use as a ventricular assist device for pediatric patients with the Inspired Universal MCS technology. The Inspired VAD Universal System is an innovative extracorporeal blood pumping system utilizing novel MagLev technology in a single portable integrated motor/controller unit which can power a variety of different disposable pump modules intended for neonate, pediatric, and adult ventricular and respiratory assistance. METHODS: A prototype of the Inspired Pediatric VAD was constructed to determine the hemodynamic requirements for pediatric applications. The magnitude/range of hydraulic torque of the internal impeller was quantified. The hydrodynamic performance of the prototype pump was benchmarked using a static mock flow loop model containing a heated blood analogue solution to test the pump over a range of rotational speeds (500-6000 RPM), flow rates (0-3.5 L/min), and pressures (0 to ~ 420 mmHg). The device was initially powered by a shaft-driven DC motor in lieu of a full MagLev design, which was also used to calculate the fluid torque acting on the impeller. RESULTS: The pediatric VAD produced flows as high as 4.27 L/min against a pressure of 127 mmHg at 6000 RPM and the generated pressure and flow values fell within the desired design specifications. CONCLUSIONS: The empirically determined performance and torque values establish the requirements for the magnetically levitated motor design to be used in the Inspired Universal MagLev System. This next step in our research and development is to fabricate a fully integrated and functional magnetically levitated pump, motor and controller system that meets the product requirement specifications and achieves a state of readiness for acute ovine animal studies to verify safety and performance of the system.

Dual-Viral Transduction Utilizing Highly Efficient Retrograde Lentivirus Improves Labeling of Long Propriospinal Neurons.

The nervous system coordinates pathways and circuits to process sensory information and govern motor behaviors. Mapping these pathways is important to further understand the connectivity throughout the nervous system and is vital for developing treatments for neuronal diseases and disorders. We targeted long ascending propriospinal neurons (LAPNs) in the rat spinal cord utilizing Fluoro-Ruby (FR) [10kD rhodamine dextran amine (RDA)], and two dual-viral systems. Dual-viral tracing utilizing a retrograde adeno-associated virus (retroAAV), which confers robust labeling in the brain, resulted in a small number of LAPNs being labeled, but dual-viral tracing using a highly efficient retrograde (HiRet) lentivirus provided robust labeling similar to FR. Additionally, dual-viral tracing with HiRet lentivirus and tracing with FR may preferentially label different subpopulations of LAPNs. These data demonstrate that dual-viral tracing in the spinal cord employing a HiRet lentivirus provides robust and specific labeling of LAPNs and emphasizes the need to empirically optimize viral systems to target specific neuronal population(s).

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells.

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

Acoustofluidic-mediated molecular delivery to human T cells with a three-dimensional-printed flow chamber.

Cell-based therapies have garnered significant interest to treat cancer and other diseases. Acoustofluidic technologies are in development to improve cell therapy manufacturing by facilitating rapid molecular delivery across the plasma membrane via ultrasound and microbubbles (MBs). In this study, a three-dimensional (3D) printed acoustofluidic device was used to deliver a fluorescent molecule, calcein, to human T cells. Intracellular delivery of calcein was assessed after varying parameters such as MB face charge, MB concentration, flow channel geometry, ultrasound pressure, and delivery time point after ultrasound treatment. MBs with a cationic surface charge caused statistically significant increases in calcein delivery during acoustofluidic treatment compared to MBs with a neutral surface charge (p < 0.001). Calcein delivery was significantly higher with a concentric spiral channel geometry compared to a rectilinear channel geometry (p < 0.001). Additionally, calcein delivery was significantly enhanced at increased ultrasound pressures of 5.1 MPa compared to lower ultrasound pressures between 0-3.8 MPa (p < 0.001). These results demonstrate that a 3D-printed acoustofluidic device can significantly enhance intracellular delivery of biomolecules to T cells, which may be a viable approach to advance cell-based therapies.

Rapid detection of SARS-CoV-2 antibodies using electrochemical impedance-based detector.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was classified as a pandemic by the World Health Organization and has caused over 550,000 deaths worldwide as of July 2020. Accurate and scalable point-of-care devices would increase screening, diagnosis, and monitoring of COVID-19 patients. Here, we demonstrate rapid label-free electrochemical detection of SARS-CoV-2 antibodies using a commercially available impedance sensing platform. A 16-well plate containing sensing electrodes was pre-coated with receptor binding domain (RBD) of SARS-CoV-2 spike protein, and subsequently tested with samples of anti-SARS-CoV-2 monoclonal antibody CR3022 (0.1 µg/ml, 1.0 µg/ml, 10 µg/ml). Subsequent blinded testing was performed on six serum specimens taken from COVID-19 and non-COVID-19 patients (1:100 dilution factor). The platform was able to differentiate spikes in impedance measurements from a negative control (1% milk solution) for all CR3022 samples. Further, successful differentiation and detection of all positive clinical samples from negative control was achieved. Measured impedance values were consistent when compared to standard ELISA test results showing a strong correlation between them (R2=0.9). Detection occurs in less than five minutes and the well-based platform provides a simplified and familiar testing interface that can be readily adaptable for use in clinical settings.

Sonoporation enables high-throughput loading of trehalose into red blood cells.

Despite recent advances in biostabilization, clinical blood supplies still experience shortages and storage limitations for red blood cells (RBCs) have not yet been sufficiently addressed. Storing RBCs in a frozen or dried state is an appealing solution to address storage limitations, but many promising cryoprotectants, including the non-reducing sugar trehalose, are impermeant to mammalian cell membranes and cannot be utilized effectively using currently available compound-loading methods. We found that transient pore formation induced by ultrasound and microbubbles (sonoporation) offers an effective means of loading trehalose into RBCs to facilitate long-term storage in a frozen or desiccated state. The protective potential of trehalose loading was demonstrated by freezing processed RBCs at -1 °C/min to -80 °C, then either storing the cells at -80 °C or lyophilizing them. RBCs were either thawed or rehydrated after 42 days of storage and evaluated for membrane integrity and esterase activity to estimate recovery and cell viability. The intracellular concentration of trehalose reached 40 mM after sonoporation and over 95% of treated RBCs were recovered after loading. Loading of trehalose was sufficient to maintain RBC morphology and esterase activity in most cells during freezing (>90% RBC recovery) and to a lower degree after lyophilization and rehydration (>20% recovery). Combining sonoporation with an integrated fluidics device allowed for rapid loading of up to 70 mM trehalose into RBCs. These results demonstrate the potential of sonoporation-mediated trehalose loading to increase recovery of viable RBCs, which could lead to effective methods for long-term stabilization of RBCs.

Design and Computational Evaluation of a Pediatric MagLev Rotary Blood Pump.

Pediatric heart failure (HF) patients have been a historically underserved population for mechanical circulatory support (MCS) therapy. To address this clinical need, we are developing a low cost, universal magnetically levitated extracorporeal system with interchangeable pump heads for pediatric support. Two impeller and pump designs (pump V1 and V2) for the pediatric pump were developed using dimensional analysis techniques and classic pump theory based on defined performance criteria (generated flow, pressure, and impeller diameter). The designs were virtually constructed using computer-aided design (CAD) software and 3D flow and pressure features were analyzed using computational fluid dynamics (CFD) analysis. Simulated pump designs (V1, V2) were operated at higher rotational speeds (~5,000 revolutions per minute [RPM]) than initially estimated (4,255 RPM) to achieve the desired operational point (3.5 L/min flow at 150 mm Hg). Pump V2 outperformed V1 by generating approximately 30% higher pressures at all simulated rotational speeds and at 5% lower priming volume. Simulated hydrodynamic performance (achieved flow and pressure, hydraulic efficiency) of our pediatric pump design, featuring reduced impeller size and priming volume, compares favorably to current commercially available MCS devices.

Increased ambient noise and elevated vocal effort contribute to airborne transmission of COVID-19.

Widespread transmission of a novel coronavirus, COVID-19, has caused major public health and economic problems around the world. Significant mitigation efforts have been implemented to reduce the spread of COVID-19 but the role of ambient noise and elevated vocal effort on airborne transmission have not been widely reported. Elevated vocal effort has been shown to increase emission of potentially infectious respiratory droplets, which can remain airborne for up to several hours. Multiple confirmed clusters of COVID-19 transmission were associated with settings where elevated vocal effort is generally required for communication, often due to high ambient noise levels, including crowded bars and restaurants, meat packing facilities, and long-stay nursing homes. Clusters of COVID-19 transmission have been frequently reported in each of these settings. Therefore, analysis of COVID-19 transmission clusters in different settings should consider whether higher ambient noise levels, which are associated with increased vocal effort, may be a contributing factor in those settings. Mitigation strategies that include reduction of ambient noise, softer speech practices, and the use of technology such as microphones and speakers to decrease vocal effort will likely reduce the risk of transmitting COVID-19 or other airborne pathogens.

Effect of speech volume on respiratory emission of oral bacteria as a potential indicator of pathogen transmissibility risk.

Respiratory droplets emitted during speech can transmit oral bacteria and infectious viruses to others, including COVID-19. Loud speech can generate significantly higher numbers of potentially infectious respiratory droplets. This study assessed the effect of speech volume on respiratory emission of oral bacteria as an indicator of potential pathogen transmission risk. Loud speech (average 83 dBA, peak 94 dBA) caused significantly higher emission of oral bacteria (p = 0.004 compared to no speech) within 1 ft from the speaker. N99 respirators and simple cloth masks both significantly reduced emission of oral bacteria. This study demonstrates that loud speech without face coverings increases emission of respiratory droplets that carry oral bacteria and may also carry other pathogens such as COVID-19.

Delivery of thymoquinone to cancer cells with as1411-conjugated nanodroplets.

Systemic delivery of conventional chemotherapies can cause negative systemic toxicity, including reduced immunity and damage to organs such as the heart and kidneys-limiting the maximum dose that can be administered. Targeted therapies appear to address this problem by having a specific target while mitigating off-target effects. Biocompatible perfluorocarbon-based nanodroplet emulsions encapsulated by a phospholipid shell are in development for delivery of molecular compounds and hold promise as vehicles for targeted delivery of chemotherapeutics to tumors. When ultrasound is applied, perfluorocarbon will undergo a phase change-ultimately inducing transient perforation of the cell membrane when in close proximity, which is more commonly known as "sonoporation." Sonoporation allows enhanced intracellular delivery of molecular compounds and will reseal to encapsulate the molecular compound intracellularly. In this study, we investigated delivery of thymoquinone (TQ), a natural hydrophobic phytochemical compound with bioactivity in cancer cells. In addition, we conjugated a G-quadruplex aptamer, 'AS1411', to TQ-loaded nanodroplets and explored their effects on multiple human cancer cell lines. AS1411 binds nucleolin, which is over-expressed on the surface of cancer cells, and in addition to its tumor-targeting properties AS1411 has also been shown to induce anti-cancer effects. Thymoquinone was loaded onto AS1411-conjugated nanodroplet emulsion to assess activity against cancer cells. Confocal microscopy indicated uptake of AS1411-conjugated nanodroplets by cancer cells. Furthermore, AS1411-conjugated nanoemulsions loaded with TQ significantly enhanced cytotoxicity in cancer cells compared to free compound. These results demonstrate that AS1411 can be conjugated onto nanodroplet emulsions for targeted delivery to human cancer cells. This novel formulation offers significant potential for targeted delivery of hydrophobic chemotherapeutics to tumors for cancer treatment.

Ultrasound-induced molecular delivery to erythrocytes using a microfluidic system.

Preservation of erythrocytes in a desiccated state for storage at ambient temperature could simplify blood transfusions in austere environments, such as rural clinics, far-forward military operations, and during space travel. Currently, storage of erythrocytes is limited by a short shelf-life of 42 days at 4 °C, and long-term preservation requires a complex process that involves the addition and removal of glycerol from erythrocytes before and after storage at -80 °C, respectively. Natural compounds, such as trehalose, can protect cells in a desiccated state if they are present at sufficient levels inside the cell, but mammalian cell membranes lack transporters for this compound. To facilitate compound loading across the plasma membrane via ultrasound and microbubbles (sonoporation), a polydimethylsiloxane-based microfluidic device was developed. Delivery of fluorescein into erythrocytes was tested at various conditions to assess the effects of parameters such as ultrasound pressure, ultrasound pulse interval, microbubble dose, and flow rate. Changes in ultrasound pressure and mean flow rate caused statistically significant increases in fluorescein delivery of up to 73 ± 37% (p < 0.05) and 44 ± 33% (p < 0.01), respectively, compared to control groups, but no statistically significant differences were detected with changes in ultrasound pulse intervals. Following freeze-drying and rehydration, recovery of viable erythrocytes increased by up to 128 ± 32% after ultrasound-mediated loading of trehalose compared to control groups (p < 0.05). These results suggest that ultrasound-mediated molecular delivery in microfluidic channels may be a viable approach to process erythrocytes for long-term storage in a desiccated state at ambient temperatures.

Ultrasound and Microbubble-targeted Delivery of a microRNA Inhibitor to the Heart Suppresses Cardiac Hypertrophy and Preserves Cardiac Function.

MicroRNAs (miRs) are dysregulated in pathological left ventricular hypertrophy. AntimiR inhibition of miR-23a suppressed hypertension-induced cardiac hypertrophy in preclinical models, but clinical translation is limited by a lack of cardiac-targeted delivery systems. Ultrasound-targeted microbubble cavitation (UTMC) utilizes microbubbles as nucleic acid carriers to target delivery of molecular therapeutics to the heart. The objective of this study was to evaluate the efficacy of UTMC targeted delivery of antimiR-23a to the hearts of mice for suppression of hypertension-induced cardiac hypertrophy. Methods: Cationic lipid microbubbles were loaded with 300 pmol negative control antimiR (NC) or antimiR-23a. Mice received continuous phenylephrine infusion via implanted osmotic minipumps, then UTMC treatments with intravenously injected antimiR-loaded microbubbles 0, 3, and 7 days later. At 2 weeks, hearts were harvested and miR-23a levels were measured. Left ventricular (LV) mass and function were assessed with echocardiography. Results: UTMC treatment with antimiR-23a decreased cardiac miR-23a levels by 41 ± 8% compared to UTMC + antimiR-NC controls (p < 0.01). Furthermore, LV mass after 1 week of phenylephrine treatment was 17 ± 10% lower following UTMC + antimiR-23a treatment compared to UTMC + antimiR-NC controls (p = 0.02). At 2 weeks, fractional shortening was 23% higher in the UTMC + antimiR-23a mice compared to UTMC + antimiR-NC controls (p < 0.01). Conclusions: UTMC is an effective technique for targeted functional delivery of antimiRs to the heart causing suppression of cardiac hypertrophy and preservation of systolic function. This approach could represent a revolutionary therapy for patients suffering from pathological cardiac hypertrophy and other cardiovascular conditions.

Effect of Molecular Weight on Sonoporation-Mediated Uptake in Human Cells.

Ultrasound-induced microbubble destruction can enhance drug delivery to cells. The molecular weight of therapeutic compounds varies significantly (from <1 kDa for small molecule drugs, to 7-15 kDa for siRNAs/miRNAs, to >1000 kDa for DNA plasmids). Therefore, the objective of this study was to determine the relationship between uptake efficiency and molecular weight using equal molar concentrations. Uptake efficiency of fluorescent compounds with different molecular weights (0.3, 10 and 2000 kDa) was explored in vitro using human cardiac mesenchymal cells and breast cancer cells exposed to microbubbles and 2.5-MHz ultrasound pulses. Uptake by viable cells was quantified using flow cytometry. After correction for the fluorescence yield of each compound, there was a significant size-dependent difference in fluorescence intensity, indicating an inverse relationship between size and uptake efficiency. These results suggest that diffusion of therapeutic compounds across permeabilized cell membranes may be an important mechanism for ultrasound-mediated drug delivery.

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.

Ultrasound Targeted Microbubble Destruction-Mediated Delivery of a Transcription Factor Decoy Inhibits STAT3 Signaling and Tumor Growth.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in many cancers where it acts to promote tumor progression. A STAT3-specific transcription factor decoy has been developed to suppress STAT3 downstream signaling, but a delivery strategy is needed to improve clinical translation. Ultrasound-targeted microbubble destruction (UTMD) has been shown to enhance image-guided local delivery of molecular therapeutics to a target site. The objective of this study was to deliver STAT3 decoy to squamous cell carcinoma (SCC) tumors using UTMD to disrupt STAT3 signaling and inhibit tumor growth. Studies performed demonstrated that UTMD treatment with STAT3 decoy-loaded microbubbles inhibited STAT3 signaling in SCC cells in vitro. Studies performed in vivo demonstrated that UTMD treatment with STAT3 decoy-loaded microbubbles induced significant tumor growth inhibition (31-51% reduced tumor volume vs. controls, p < 0.05) in mice bearing SCC tumors. Furthermore, expression of STAT3 downstream target genes (Bcl-xL and cyclin D1) was significantly reduced (34-39%, p < 0.05) in tumors receiving UTMD treatment with STAT3 decoy-loaded microbubbles compared to controls. In addition, the quantity of radiolabeled STAT3 decoy detected in tumors eight hours after treatment was significantly higher with UTMD treatment compared to controls (70-150%, p < 0.05). This study demonstrates that UTMD can increase delivery of a transcription factor decoy to tumors in vivo and that the decoy can inhibit STAT3 signaling and tumor growth. These results suggest that UTMD treatment holds potential for clinical use to increase the concentration of a transcription factor signaling inhibitor in the tumor.

Microfluidic fabrication of perfluorohexane-shelled double emulsions for controlled loading and acoustic-triggered release of hydrophilic agents.

The ability of low boiling point liquid perfluorocarbons (PFCs) to undergo a phase change from a liquid to a gas upon ultrasound irradiation makes PFC-based emulsions promising vehicles for triggered delivery of payloads. However, loading hydrophilic agents into PFC-based emulsions is difficult due to their insolubility in PFC. Here, we address this challenge by taking advantage of microfluidic technologies to fabricate double emulsions consisting of large aqueous cores and a perfluorohexane (PFH) shell, thus yielding high loading capacities for hydrophilic agents. Using this technology, we efficiently encapsulate a model hydrophilic agent within the emulsions and study its response to ultrasound irradiation. Using a combination of optical and acoustic imaging methods, we observe payload release upon acoustic vaporization of PFH. Our work demonstrates the utility of microfluidic techniques for controllably loading hydrophilic agents into PFH-based emulsions, which have great potential for acoustically triggered release.

Acoustic characterization of monodisperse lipid-coated microbubbles: relationship between size and shell viscoelastic properties.

The acoustic attenuation spectrum of lipid-coated microbubble suspensions was measured in order to characterize the linear acoustic behavior of ultrasound contrast agents. For that purpose, microbubbles samples were generated with a very narrow size distribution by using microfluidics techniques. A performance as good as optical characterization techniques of single microbubbles was achieved using this method. Compared to polydispersions (i.e., contrast agents used clinically), monodisperse contrast agents have a narrower attenuation spectrum, which presents a maximum peak at a frequency value corresponding to the average single bubble resonance frequency. The low polydispersity index of the samples made the estimation of the lipid viscoelastic properties more accurate since, as previously reported, the shell linear parameters may change with the equilibrium bubble radius. The results showed the great advantage of dealing with monodisperse populations rather than polydisperse populations for the acoustic characterization of ultrasound contrast agents.