Ultrasound enhanced siRNA delivery using cationic liposome-microbubble complexes for the treatment of squamous cell carcinoma.

Rationale: Microbubble (MB) contrast agents combined with ultrasound targeted microbubble cavitation (UTMC) are a promising platform for site-specific therapeutic oligonucleotide delivery. We investigated UTMC-mediated delivery of siRNA directed against epidermal growth factor receptor (EGFR), to squamous cell carcinoma (SCC) via a novel MB-liposome complex (LPX). Methods: LPXs were constructed by conjugation of cationic liposomes to the surface of C4F10 gas-filled lipid MBs using biotin/avidin chemistry, then loaded with siRNA via electrostatic interaction. Luciferase-expressing SCC-VII cells (SCC-VII-Luc) were cultured in Petri dishes. The Petri dishes were filled with media in which LPXs loaded with siRNA against firefly luciferase (Luc siRNA) were suspended. Ultrasound (US) (1 MHz, 100-µs pulse, 10% duty cycle) was delivered to the dishes for 10 sec at varying acoustic pressures and luciferase assay was performed 24 hr later. In vivo siRNA delivery was studied in SCC-VII tumor-bearing mice intravenously infused with a 0.5 mL saline suspension of EGFR siRNA LPX (7×108 LPX, ~30 µg siRNA) for 20 min during concurrent US (1 MHz, 0.5 MPa spatial peak temporal peak negative pressure, five 100-µs pulses every 1 ms; each pulse train repeated every 2 sec to allow reperfusion of LPX into the tumor). Mice were sacrificed 2 days post treatment and tumor EGFR expression was measured (Western blot). Other mice (n=23) received either EGFR siRNA-loaded LPX + UTMC or negative control (NC) siRNA-loaded LPX + UTMC on days 0 and 3, or no treatment ("sham"). Tumor volume was serially measured by high-resolution 3D US imaging. Results: Luc siRNA LPX + UTMC caused significant luciferase knockdown vs. no treatment control, p<0.05) in SCC-VII-Luc cells at acoustic pressures 0.25 MPa to 0.9 MPa, while no significant silencing effect was seen at lower pressure (0.125 MPa). In vivo, EGFR siRNA LPX + UTMC reduced tumor EGFR expression by ~30% and significantly inhibited tumor growth by day 9 (~40% decrease in tumor volume vs. NC siRNA LPX + UTMC, p<0.05). Conclusions: Luc siRNA LPXs + UTMC achieved functional delivery of Luc siRNA to SCC-VII-Luc cells in vitro. EGFR siRNA LPX + UTMC inhibited tumor growth and suppressed EGFR expression in vivo, suggesting that this platform holds promise for non-invasive, image-guided targeted delivery of therapeutic siRNA for cancer treatment.

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.

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.

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.

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.

Cavitation-enhanced MR-guided focused ultrasound ablation of rabbit tumors in vivo using phase shift nanoemulsions.

Advanced tumors are often inoperable due to their size and proximity to critical vascular structures. High intensity focused ultrasound (HIFU) has been developed to non-invasively thermally ablate inoperable solid tumors. However, the clinical feasibility of HIFU ablation therapy has been limited by the long treatment times (on the order of hours) and high acoustic intensities required. Studies have shown that inertial cavitation can enhance HIFU-mediated heating by generating broadband acoustic emissions that increase tissue absorption and accelerate HIFU-induced heating. Unfortunately, initiating inertial cavitation in tumors requires high intensities and can be unpredictable. To address this need, phase-shift nanoemulsions (PSNE) have been developed. PSNE consist of lipid-coated liquid perfluorocarbon droplets that are less than 200 nm in diameter, thereby allowing passive accumulation in tumors through leaky tumor vasculature. PSNE can be vaporized into microbubbles in tumors in order to nucleate cavitation activity and enhance HIFU-mediated heating. In this study, MR-guided HIFU treatments were performed on intramuscular rabbit VX2 tumors in vivo to assess the effect of vaporized PSNE on acoustic cavitation and HIFU-mediated heating. HIFU pulses were delivered for 30 s using a 1.5 MHz, MR-compatible transducer, and cavitation emissions were recorded with a 650 kHz ring hydrophone while temperature was monitored using MR thermometry. Cavitation emissions were significantly higher (P < 0.05) after PSNE injection and this was well correlated with enhanced HIFU-mediated heating in tumors. The peak temperature rise induced by sonication was significantly higher (P < 0.05) after PSNE injection. For example, the mean per cent change in temperature achieved at 5.2 W of acoustic power was 46 ± 22% with PSNE injection. The results indicate that PSNE nucleates cavitation which correlates with enhanced HIFU-mediated heating in tumors. This suggests that PSNE could potentially be used to reduce the time and/or acoustic intensity required for HIFU-mediated heating, thereby increasing the feasibility and clinical efficacy of HIFU thermal ablation therapy.

Triggered drug release from superhydrophobic meshes using high-intensity focused ultrasound.

Application of high-intensity focused ultrasound to drug-loaded superhydrophobic meshes affords triggered drug release by displacing an entrapped air layer. The air layer within the superhydrophobic meshes is characterized using direct visualization and B-mode imaging. Drug-loaded superhydrophobic meshes are cytotoxic in an in vitro assay after ultrasound treatment.

Accumulation of phase-shift nanoemulsions to enhance MR-guided ultrasound-mediated tumor ablation in vivo.

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is being explored as a non-invasive technology to treat solid tumors. However, the clinical use of HIFU for tumor ablation applications is currently limited by the long treatment times required. Phase-shift nanoemulsions (PSNE), consisting of liquid perfluorocarbon droplets that can be vaporized into microbubbles, are being developed to accelerate HIFU-mediated heating. The purpose of this study was to examine accumulation of PSNE in intramuscular rabbit tumors in vivo. MR images were acquired before and after intravenous injection of gadolinium-containing PSNE. MR signal enhancement was observed in rabbit tumors up to six hours after injection, indicating that PSNE accumulated in the tumors. In addition, PSNE vaporization was detected in the tumor with B-mode ultrasound imaging, and MR thermometry measurements indicated that PSNE accelerated the rate of HIFU-mediated heating. These results suggest that PSNE could dramatically improve the efficiency and clinical feasibility of MRgHIFU.

The impact of vaporized nanoemulsions on ultrasound-mediated ablation.

BACKGROUND: The clinical feasibility of using high-intensity focused ultrasound (HIFU) for ablation of solid tumors is limited by the high acoustic pressures and long treatment times required. The presence of microbubbles during sonication can increase the absorption of acoustic energy and accelerate heating. However, formation of microbubbles within the tumor tissue remains a challenge. Phase-shift nanoemulsions (PSNE) have been developed as a means for producing microbubbles within tumors. PSNE are emulsions of submicron-sized, lipid-coated, and liquid perfluorocarbon droplets that can be vaporized into microbubbles using short (<1 ms), high-amplitude (>5 MPa) acoustic pulses. In this study, the impact of vaporized phase-shift nanoemulsions on the time and acoustic power required for HIFU-mediated thermal lesion formation was investigated in vitro. METHODS: PSNE containing dodecafluoropentane were produced with narrow size distributions and mean diameters below 200 nm using a combination of sonication and extrusion. PSNE was dispersed in albumin-containing polyacrylamide gel phantoms for experimental tests. Albumin denatures and becomes opaque at temperatures above 58°C, enabling visual detection of lesions formed from denatured albumin. PSNE were vaporized using a 30-cycle, 3.2-MHz, at an acoustic power of 6.4 W (free-field intensity of 4,586 W/cm(2)) pulse from a single-element, focused high-power transducer. The vaporization pulse was immediately followed by a 15-s continuous wave, 3.2-MHz signal to induce ultrasound-mediated heating. Control experiments were conducted using an identical procedure without the vaporization pulse. Lesion formation was detected by acquiring video frames during sonication and post-processing the images for analysis. Broadband emissions from inertial cavitation (IC) were passively detected with a focused, 2-MHz transducer. Temperature measurements were acquired using a needle thermocouple. RESULTS: Bubbles formed at the HIFU focus via PSNE vaporization enhanced HIFU-mediated heating. Broadband emissions detected during HIFU exposure coincided in time with measured accelerated heating, which suggested that IC played an important role in bubble-enhanced heating. In the presence of bubbles, the acoustic power required for the formation of a 9-mm(3) lesion was reduced by 72% and the exposure time required for the onset of albumin denaturation was significantly reduced (by 4 s), provided that the PSNE volume fraction in the polyacrylamide gel was at least 0.008%. CONCLUSIONS: The time or acoustic power required for lesion formation in gel phantoms was dramatically reduced by vaporizing PSNE into bubbles. These results suggest that PSNE may improve the efficiency of HIFU-mediated thermal ablation of solid tumors; thus, further investigation is warranted to determine whether bubble-enhanced HIFU may potentially become a viable option for cancer therapy.

Synthesis of phase-shift nanoemulsions with narrow size distributions for acoustic droplet vaporization and bubble-enhanced ultrasound-mediated ablation.

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.

Delivery of stem cells to porcine arterial wall with echogenic liposomes conjugated to antibodies against CD34 and intercellular adhesion molecule-1.

In atherosclerosis, the loss of vascular stem cells via apoptosis impairs the capacity of the vascular wall to repair or regenerate the tissue damaged by atherogenic factors. Recruitment of exogenous stem cells to the plaque tissue may repopulate vascular cells and help repair the arterial tissue. Ultrasound-enhanced liposomal targeting may provide a feasible method for stem cell delivery into atheroma. Bifunctional echogenic immunoliposomes (BF-ELIP) were generated by covalently coupling two antibodies to liposomes; the first one specific for CD34 antigens on the surface of stem cells and the second directed against the intercellular adhesion molecule-1 (ICAM-1) antigens on the inflammatory endothelium covering atheroma. CD34+ stem cells from adult bone marrow were incubated on the ICAM-1-expressing endothelium of the aorta of swine fed high cholesterol diets, which was preloaded with BF-ELIP. Significantly increased stem cell adherence and penetration were detected in particular in the aortic segments treated with 1 MHz low-amplitude continuous wave ultrasound. Fluorescence and scanning electron microscopy confirmed the presence of BF-ELIP-bound CD34+ cells in the intimal compartment of the atheromatous arterial wall. Ultrasound treatment increased the number of endothelial cell progenitors migrating into the intima. Thus, under ultrasound enhancement, BF-ELIP bound CD34+ stem cells selectively bind to the ICAM-1 expressing endothelium of atherosclerotic lesions.