Naomi Matsuura

Vaporization of perfluorocarbon droplets using optical irradiation.

Micron-sized liquid perfluorocarbon (PFC) droplets are currently being investigated as activatable agents for medical imaging and cancer therapy. After injection into the bloodstream, superheated PFC droplets can be vaporized to a gas phase for ultrasound imaging, or for cancer therapy via targeted drug delivery and vessel occlusion. Droplet vaporization has been previously demonstrated using acoustic methods. We propose using laser irradiation as a means to induce PFC droplet vaporization using a method we term optical droplet vaporization (ODV). In order to facilitate ODV of PFC droplets which have negligible absorption in the infrared spectrum, optical absorbing nanoparticles were incorporated into the droplet. In this study, micron-sized PFC droplets loaded with silica-coated lead sulfide (PbS) nanoparticles were evaluated using a 1064 nm laser and ultra-high frequency photoacoustic ultrasound (at 200 and 375 MHz). The photoacoustic response was proportional to nanoparticle loading and successful optical droplet vaporization of individual PFC droplets was confirmed using photoacoustic, acoustic, and optical measurements. A minimum laser fluence of 1.4 J/cm(2) was required to vaporize the droplets. The vaporization of PFC droplets via laser irradiation can lead to the activation of PFC agents in tissues previously not accessible using standard ultrasound-based techniques.

Microfluidic Assembly of Monodisperse, Nanoparticle-Incorporated Perfluorocarbon Microbubbles for Medical Imaging and Therapy

New medical imaging contrast agents that permit multiple imaging and therapy applications using a single agent can result in more accurate diagnosis and local treatment of diseased tissue. Solid nanoparticles (NPs) (5-150 nm in size) have emerged as promising imaging and therapy agents, as have micrometer-scale, perfluorocarbon gas-filled microbubbles (MBs) used in patients as intravascular ultrasound contrast agents. We propose that the modular combination of small, solid NPs and larger, highly compressible MBs into a single agent is an effective way to attain the desired complementary and hybrid properties of two very different agents. Presented here is a new strategy for the simple and robust incorporation of various medical NPs with monodisperse MBs based upon the controlled pH-based regulation of the electrostatic attraction between NPs and the MB shell. Using this simple approach, microfluidic-generated, protein-lipid-coated, perfluorobutane MBs (with size control down to 3 microm) were incorporated with silica-coated NPs, including CdSe/ZnS quantum dots, gold nanorods, iron oxide NPs, and Gd-loaded mesoporous silica NPs. The silica interface permits NP inclusion within MBs to be independent of NP composition, morphology, and size. Significantly, the NP-incorporated MBs (NP-MBs) diluted in saline were detectable using low-pressure ultrasound, and the monodisperse MB platform can be produced at high-throughput, sufficient for in vivo usage (10(6) MB/sec). The modular synthesis of a variety of NP-MBs can facilitate flexible, user-defined, multifunctional imaging and therapy agents tailored for specific applications and disease types.

Digital radiology using active matrix readout: amplified pixel detector array for fluoroscopy.

Active matrix array technology has made possible the concept of flat panel imaging systems for radiography. In the conventional approach a thin-film circuit built on glass contains the necessary switching components (thin-film transistors or TFTs) to readout an image formed in either a phosphor or photoconductor layer. Extension of this concept to real time imaging--fluoroscopy--has had problems due to the very low noise required. A new design strategy for fluoroscopic active matrix flat panel detectors has therefore been investigated theoretically. In this approach, the active matrix has integrated thin-film amplifiers and readout electronics at each pixel and is called the amplified pixel detector array (APDA). Each amplified pixel consists of three thin-film transistors: an amplifier, a readout, and a reset TFT. The performance of the APDA approach compared to the conventional active matrix was investigated for two semiconductors commonly used to construct active matrix arrays--hydrogenated amorphous silicon and polycrystalline silicon. The results showed that with amplification close to the pixel, the noise from the external charge preamplifiers becomes insignificant. The thermal and flicker noise of the readout and the amplifying TFTs at the pixel become the dominant sources of noise. The magnitude of these noise sources is strongly dependent on the TFT geometry and its fabrication process. Both of these could be optimized to make the APDA active matrix operate at lower noise levels than is possible with the conventional approach. However, the APDA cannot be made to operate ideally (i.e., have noise limited only by the amount of radiation used) at the lowest exposure rate required in medical fluoroscopy.

Optical studies of vaporization and stability of fluorescently labelled perfluorocarbon droplets

Droplets of liquid perfluorocarbon (PFC) are under study as the next generation of contrast agents for ultrasound (US). These droplets can be selectively vaporized into echogenic gas bubbles in situ by externally applied US, with numerous applications to diagnosis and therapy. However, little is known about the mechanisms of droplet vaporization and the stability of the bubbles so produced. Here we observe optically the vaporization of fluorescent PFC droplets and the stability of the newly created bubbles. Fluorescent markers were used to label selectively either the liquid PFC core or the shell of the droplets. It was found that, following vaporization, the fluorescent marker is quickly expelled from the core of the newly created bubble and is retained on the gas-liquid interface. At the same time, it was shown that bubbles retain the original shells encapsulating their droplet precursors. The efficiency of encapsulation was found to depend strongly on the nature of the stabilizing material itself. These results provide direct evidence of droplet encapsulation post-vaporization, and suggest that the behaviour of the vaporized droplets is strongly dependent on the choice of the stabilizing material for the emulsion.

Towards new functional nanostructures for medical imaging

Nanostructures represent a promising new type of contrast agent for clinical medical imaging modalities, including magnetic resonance imaging, x-ray computed tomography, ultrasound, and nuclear imaging. Currently, most nanostructures are simple, single-purpose imaging agents based on spherical constructs (e.g., liposomes, micelles, nanoemulsions, macromolecules, dendrimers, and solid nanoparticle structures). In the next decade, new clinical imaging nanostructures will be designed as multi-functional constructs, to both amplify imaging signals at disease sites and deliver localized therapy. Proposals for nanostructures to fulfill these new functions will be outlined. New functional nanostructures are expected to develop in five main directions: Modular nanostructures with additive functionality; cooperative nanostructures with synergistic functionality; nanostructures activated by their in vivo environment; nanostructures activated by sources outside the patient; and novel, nonspherical nanostructures and components. The development and clinical translation of next-generation nanostructures will be facilitated by a combination of improved clarity of the in vivo imaging and biological challenges and the requirements to successfully overcome them; development of standardized characterization and validation systems tailored for the preclinical assessment of nanostructure agents; and development of streamlined commercialization strategies and pipelines tailored for nanostructure-based agents for their efficient translation to the clinic.

Acoustic and photoacoustic characterization of micron-sized perfluorocarbon emulsions

Abstract. Perfluorocarbon droplets containing nanoparticles (NPs) have recently been investigated as theranostic and dual-mode contrast agents. These droplets can be vaporized via laser irradiation or used as photoacoustic contrast agents below the vaporization threshold. This study investigates the photoacoustic mechanism of NP-loaded droplets using photoacoustic frequencies between 100 and 1000 MHz, where distinct spectral features are observed that are related to the droplet composition. The measured photoacoustic spectrum from NP-loaded perfluorocarbon droplets was compared to a theoretical model that assumes a homogenous liquid. Good agreement in the location of the spectral features was observed, which suggests the NPs act primarily as optical absorbers to induce thermal expansion of the droplet as a single homogenous object. The NP size and composition do not affect the photoacoustic spectrum; therefore, the photoacoustic signal can be maximized by optimizing the NP optical absorbing properties. To confirm the theoretical parameters in the model, photoacoustic, ultrasonic, and optical methods were used to estimate the droplet diameter. Photoacoustic and ultrasonic methods agreed to within 1.4%, while the optical measurement was 8.5% higher; this difference decreased with increasing droplet size. The small discrepancy may be attributed to the difficulty in observing the small droplets through the partially translucent phantom.

Silica-coated quantum dots for optical evaluation of perfluorocarbon droplet interactions with cells.

There has been recent interest in developing new, targeted, perfluorocarbon (PFC) droplet-based contrast agents for medical imaging (e.g., magnetic resonance imaging, X-ray/computed tomography, and ultrasound imaging). However, due to the large number of potential PFCs and droplet stabilization strategies available, it is challenging to determine in advance the PFC droplet formulation that will result in the optimal in vivo behavior and imaging performance required for clinical success. We propose that the integration of fluorescent quantum dots (QDs) into new PFC droplet agents can help to rapidly screen new PFC-based candidate agents for biological compatibility early in their development. QD labels can allow the interaction of PFC droplets with single cells to be assessed at high sensitivity and resolution using optical methods in vitro, complementing the deeper depth penetration but lower resolution provided by PFC droplet imaging using in vivo medical imaging systems. In this work, we introduce a simple and robust method to miscibilize silica-coated nanoparticles into hydrophobic and lipophobic PFCs through fluorination of the silica surface via a hydrolysis-condensation reaction with 1H,1H,2H,2H-perfluorodecyltriethoxysilane. Using CdSe/ZnS core/shell QDs, we show that nanoscale, QD-labeled PFC droplets can be easily formed, with similar sizes and surface charges as unlabeled PFC droplets. The QD label can be used to determine the PFC droplet uptake into cells in vitro by fluorescence microscopy and flow cytometry, and can be used to validate the fate of PFC droplets in vivo in small animals via fluorescence microscopy of histological tissue sections. This is demonstrated in macrophage and cancer cells, and in rabbits, respectively. This work reveals the potential of using QD labels for rapid, preclinical, optical assessment of different PFC droplet formulations for their future use in patients.

Modeling photoacoustic spectral features of micron-sized particles

The photoacoustic signal generated from particles when irradiated by light is determined by attributes of the particle such as the size, speed of sound, morphology and the optical absorption coefficient. Unique features such as periodically varying minima and maxima are observed throughout the photoacoustic signal power spectrum, where the periodicity depends on these physical attributes. The frequency content of the photoacoustic signals can be used to obtain the physical attributes of unknown particles by comparison to analytical solutions of homogeneous symmetric geometric structures, such as spheres. However, analytical solutions do not exist for irregularly shaped particles, inhomogeneous particles or particles near structures. A finite element model (FEM) was used to simulate photoacoustic wave propagation from four different particle configurations: a homogeneous particle suspended in water, a homogeneous particle on a reflecting boundary, an inhomogeneous particle with an absorbing shell and non-absorbing core, and an irregularly shaped particle such as a red blood cell. Biocompatible perfluorocarbon droplets, 3-5 μm in diameter containing optically absorbing nanoparticles were used as the representative ideal particles, as they are spherical, homogeneous, optically translucent, and have known physical properties. The photoacoustic spectrum of micron-sized single droplets in suspension and on a reflecting boundary were measured over the frequency range of 100-500 MHz and compared directly to analytical models and the FEM. Good agreement between the analytical model, FEM and measured values were observed for a droplet in suspension, where the spectral minima agreed to within a 3.3 MHz standard deviation. For a droplet on a reflecting boundary, spectral features were correctly reproduced using the FEM but not the analytical model. The photoacoustic spectra from other common particle configurations such as particle with an absorbing shell and a biconcave-shaped red blood cell were also investigated, where unique features in the power spectrum could be used to identify them.

Ultrahigh-density, nonlithographic, sub-100 nm pattern transfer by ion implantation and selective chemical etching

A self-assembled array of nanometer-sized holes in alumina has been adapted as a mask for conventional, broad-area, ion implantation. The mask pattern, made up of nanoholes arranged in a two-dimensional triangular array with a 100 nm period and a 55 nm diameter pore size, has been successfully transferred onto single crystal (100) SrTiO3 substrates using 200 and 500 keV energy Pt ion bombardments, at fluences sufficient to amorphize the exposed areas. The amorphized material was removed by selective chemical etching resulting in a periodic array of holes about 55 nm in diameter and 115 nm deep. This parallel, nonlithographic approach is adaptable to submicron depth, variable array geometry and scale, and to any material where a selective etch can be found for the irradiated volume.

Nanoparticle-loaded perfluorocarbon droplets for imaging and therapy

Nanoscale perfluorocarbon droplets that are in the liquid phase at physiological temperatures, but which can be converted to gas using ultrasound, offer potential as a contrast agent for the detection and therapy of solid tumours. Nanoparticles such as quantum dots can also be encapsulated within PFC droplets, enabling multi-modal imaging and controlled nanoparticle release. In this work, experiments were conducted to investigate the impact of nanoparticle incorporation on droplet conversion at low and high ultrasound frequencies. It was found that incorporation of quantum dots lowered the inertial cavitation threshold at 1 MHz by 20%. In contrast, quantum dot nanoparticles did not significantly alter the conversion threshold of perfluorohexane or perfluoropentane droplets at 18 MHz. It was also shown that perfluoropentane droplets could be converted to gas and imaged at high frequency in hepatomas in mice, using brief high pressure bursts to achieve the phase conversion. Finally, optically fluorescent quantum dots incorporated within droplets were used to demonstate the feasibility of assessing biodistribution in rabbits using fluorescence histology.