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Magnetic nanodroplets for targeted drug delivery
Abstracts Sonofluidic fabrication of microbubbles for imaging and therapy Elanor Stride Professor of Engineering Science, University of Oxford, Oxford, United Kingdom Gas-filled microbubbles are routinely used in diagnostic ultrasound imaging as contrast agents, and are under investigation for therapeutic application including drug delivery and ablation enhancement. They typically consist of a high molecular weight gas surrounded by a coating of surfactant, denatured albumin and/or a polymer. The acoustic response of microbubbles is profoundly influenced by their physical characteristics, in particular their size, size distribution, and the rheological properties of the coating. These in turn depend on the chemical formulation of the microbubble shell and the production technique. Sonication is the most commonly employed method and can generate high concentrations of microbubbles rapidly but with a broad size distribution and wide variability in acoustic response. Microfluidic devices provide excellent control over size, but the small-scale architectures required are often challenging to manufacture, offer low production rates, and are prone to clogging. Microfluidic generated microbubbles may also have inferior surface characteristics and stability compared to those produced by sonication. In order to address the limitations of the existing methods, we have developed a hybrid “sonofluidic” device. Monodisperse bubbles of a few 100 mm in diameter are first produced using a simple T-junction with relatively large channel dimensions (254mm x 50mm). Consequently high flow rates can be used with minimal risk of clogging or leakage. After formation, the large bubbles are exposed to ultrasound from a transducer embedded within the device for 1s over a frequency range of 71-73kHz. This promotes controlled fragmentation of the large bubbles to generate bubbles of the size required for clinical applications. Microbubbles were prepared using the sonofluidic device, a conventional microfluidic system or a standard sonication protocol. They were compared in terms of their size, size distribution, concentration, stability, acoustic response, and surface molecular concentration using quantitative fluorescence microscopy. The characteristics of the microbubbles produced by the sonofluidic device were found to be equivalent in terms of production rate and stability to those formed by sonication; but to have a narrower size distribution, closer to that obtained with microfluidics. These differences were reflected in the measured acoustic response and surface properties.
Magnetic nanodroplets for targeted drug delivery Elanor Stride Professor of Engineering Science, University of Oxford, Oxford, United Kingdom A limitation of microbubbles for both diagnostic and therapeutic applications is their relatively rapid clearance times, typically < 10 minutes. One solution to this is to use nanoscale droplets of volatile liquids that can be converted into microbubbles upon exposure to ultrasound. Their small size both significantly enhances their circulation time and also enables them to extravasate, for example in the leaky vasculature within a tumour. Notwithstanding their significant potential, the development of nanodroplets still poses some considerable challenges. The conversion efficiency, i.e., the proportion of droplets undergoing a phase change for a given set of ultrasound exposure conditions is often very low. Thus either very high concentrations of nanodroplets or potentially damaging ultrasound intensities are required. We therefore investigated the effect of loading perfluorocarbon nanodroplets with superparamagnetic solid nanoparticles to act as nucleation agents to promote phase transition thereby improving conversion
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efficiency. Using iron oxide particles also provides a means of imaging the droplets using magnetic resonance imaging (MRI) and manipulating the droplets using an external magnetic field which has been shown in previous work to be highly advantageous for drug delivery. We first determined the effect upon the physical properties of the nanodroplets in terms of their size, surface charge, and stability under physiological conditions. Their response to ultrasound exposure was then examined to determine conversion efficiency, change in size and potential for image contrast enhancement under both ultrasound and MRI. Finally, delivery of a small molecule chemotherapy drug, paclitaxel, and small interfering ribonucleic acid (siRNA) to different cancer cell lines was investigated. The nanodroplets were stable at 37˚C over 10 days and showed a significantly higher conversion efficiency than a control droplet formulated without nanoparticles at 1 MHz and peak negative pressures < 500 kPa. They could be readily imaged at clinically safe concentrations using MRI and ultrasound and targeted using an externally applied magnetic field. Successful delivery of both paclitaxel and siRNA was demonstrated, with higher rates of cell death and transfection respectively achieved than with the control formulation. Magnetic nanodroplets for targeted drug delivery
Acoustic protein nanostructures for contrast ultrasound F. Stuart Foster, Judy Yan, Yohannes Soenjaya, Christine E.M. Demore Sunnybrook Research Institute and Department of Medical Biophysics, University of Toronto,Toronto, ON, Canada Gas vesicles (GVs) are nanosized protein-encased gas particles produced in species of Bacteria and Archea. Similar to microbubbles, GVs can generate harmonic signals when exposed to ultrasound, making it an alternative agent for contrast imaging. In addition, their protein shell allows for continuous gas exchange with the environment resulting in a physically stable long-lasting structure. A further advantage of gas vesicles is their size (40-500 nm), which has the potential for extravasation out of leaky vasculatures of tumors. This can potentially allow for targeting molecular biomarkers outside of the vasculature. Using the VevoÒ 2100 system (FUJIFILM VisualSonics, Toronto, Canada) operating at 18MHz, we imaged mouse tumor models to optimize and improve signal enhancement. GVs were isolated and purified from Halobacterium sp. NRC-1 and quantified using the optical density at 500nm (OD500). Mouse tumor models were generated using Lewis Lung Carcinoma cells injected into SHO mice and imaged after 14 days. A total of 300 mL of GVs at a concentration of 40 OD500 was injected intravenously at an infusion rate of 500 mL/min. Non-linear contrast enhancement in tumors was clearly visible after infusion of GVs, with wash-in curves showing high SNR. Unfortunately, in contrast to the liver where a prolong enhancement was observed, a rapid loss of contrast was seen in tumors post-injection. Inhibition of the RES is a well-known method to improve circulation of nanosized particles. The saturation of liver Kupffer cells using a double injection showed better signal retention (40-70%) increase following the second administration of GVs, in comparison to the first. Additionally, the disruption of Kupffer cell function using either GdCl3 or Intralipid also resulted in prolonged signal retention. To examine a more biocompatible method of blocking GVs detection by the RES, the surface of GVs were modified with the addition of PEG chains of varying lengths. A 5 kDa PEG length showed 75% signal retention during the washout phase of contrast enhancement. References: 1. Shapiro MG, et al. Nat Nanotechnol 9, 311-6 (2014).
Magnetic nanodroplets for targeted drug delivery Elanor Stride Professor of Engineering Science, University of Oxford, Oxford, United Kingdom A limitation of microbubbles for both diagnostic and therapeutic applications is their relatively rapid clearance times, typically < 10 minutes. One solution to this is to use nanoscale droplets of volatile liquids that can be converted into microbubbles upon exposure to ultrasound. Their small size both significantly enhances their circulation time and also enables them to extravasate, for example in the leaky vasculature within a tumour. Notwithstanding their significant potential, the development of nanodroplets still poses some considerable challenges. The conversion efficiency, i.e., the proportion of droplets undergoing a phase change for a given set of ultrasound exposure conditions is often very low. Thus either very high concentrations of nanodroplets or potentially damaging ultrasound intensities are required. We therefore investigated the effect of loading perfluorocarbon nanodroplets with superparamagnetic solid nanoparticles to act as nucleation agents to promote phase transition thereby improving conversion
S49
efficiency. Using iron oxide particles also provides a means of imaging the droplets using magnetic resonance imaging (MRI) and manipulating the droplets using an external magnetic field which has been shown in previous work to be highly advantageous for drug delivery. We first determined the effect upon the physical properties of the nanodroplets in terms of their size, surface charge, and stability under physiological conditions. Their response to ultrasound exposure was then examined to determine conversion efficiency, change in size and potential for image contrast enhancement under both ultrasound and MRI. Finally, delivery of a small molecule chemotherapy drug, paclitaxel, and small interfering ribonucleic acid (siRNA) to different cancer cell lines was investigated. The nanodroplets were stable at 37˚C over 10 days and showed a significantly higher conversion efficiency than a control droplet formulated without nanoparticles at 1 MHz and peak negative pressures < 500 kPa. They could be readily imaged at clinically safe concentrations using MRI and ultrasound and targeted using an externally applied magnetic field. Successful delivery of both paclitaxel and siRNA was demonstrated, with higher rates of cell death and transfection respectively achieved than with the control formulation. Magnetic nanodroplets for targeted drug delivery
Acoustic protein nanostructures for contrast ultrasound F. Stuart Foster, Judy Yan, Yohannes Soenjaya, Christine E.M. Demore Sunnybrook Research Institute and Department of Medical Biophysics, University of Toronto,Toronto, ON, Canada Gas vesicles (GVs) are nanosized protein-encased gas particles produced in species of Bacteria and Archea. Similar to microbubbles, GVs can generate harmonic signals when exposed to ultrasound, making it an alternative agent for contrast imaging. In addition, their protein shell allows for continuous gas exchange with the environment resulting in a physically stable long-lasting structure. A further advantage of gas vesicles is their size (40-500 nm), which has the potential for extravasation out of leaky vasculatures of tumors. This can potentially allow for targeting molecular biomarkers outside of the vasculature. Using the VevoÒ 2100 system (FUJIFILM VisualSonics, Toronto, Canada) operating at 18MHz, we imaged mouse tumor models to optimize and improve signal enhancement. GVs were isolated and purified from Halobacterium sp. NRC-1 and quantified using the optical density at 500nm (OD500). Mouse tumor models were generated using Lewis Lung Carcinoma cells injected into SHO mice and imaged after 14 days. A total of 300 mL of GVs at a concentration of 40 OD500 was injected intravenously at an infusion rate of 500 mL/min. Non-linear contrast enhancement in tumors was clearly visible after infusion of GVs, with wash-in curves showing high SNR. Unfortunately, in contrast to the liver where a prolong enhancement was observed, a rapid loss of contrast was seen in tumors post-injection. Inhibition of the RES is a well-known method to improve circulation of nanosized particles. The saturation of liver Kupffer cells using a double injection showed better signal retention (40-70%) increase following the second administration of GVs, in comparison to the first. Additionally, the disruption of Kupffer cell function using either GdCl3 or Intralipid also resulted in prolonged signal retention. To examine a more biocompatible method of blocking GVs detection by the RES, the surface of GVs were modified with the addition of PEG chains of varying lengths. A 5 kDa PEG length showed 75% signal retention during the washout phase of contrast enhancement. References: 1. Shapiro MG, et al. Nat Nanotechnol 9, 311-6 (2014).