18F-Labeled perfluorocarbon droplets for positron emission tomography imaging.

INTRODUCTION: Nanoscale perfluorocarbon (PFC) droplets have been used to create imaging agents and drug delivery vehicles. However, development and characterization of new formulations of PFC droplets are hindered because of the lack of simple methods for quantitative and sensitive assessment of whole body tissue distribution and pharmacokinetics of the droplets. To address this issue, a general-purpose method for radiolabeling the inner core of nanoscale perfluorocarbon droplets with a hydrophobic and lipophobic fluorine-18 compound was developed, so that positron emission tomography (PET) and quantitative biodistribution studies can be employed to evaluate PFC nanodroplets in vivo. METHODS: A robust method to produce [18F]CF3(CF2)7(CH2)3F from a tosylate precursor using [18F]F- was developed. The product's effectiveness as a general label for different PFCs and its ability to distinguish the in vivo behavior of different PFC droplet formulations was evaluated using two types of PFC nanodroplets: fluorosurfactant-stabilized perfluorohexane (PFH) nanodroplets and lipid-stabilized perfluorooctylbromide (PFOB) nanodroplets. In vivo assessment of the 18F-labeled PFH and PFOB nanodroplets were conducted in normal mice following intravenous injection using small animal PET imaging and gamma counting of tissues and fluids. RESULTS: [18F]CF3(CF2)7(CH2)3F was produced in modest yield and was stable with respect to loss of fluoride in vitro. The labeled fluorocarbon was successfully integrated into PFH nanodroplets (~175 nm) and PFOB nanodroplets (~260 nm) without altering their mean sizes, size distributions, or surface charges compared to their non-radioactive analogues. No leakage of the radiolabel from the nanodroplets was detected after droplet formation in vitro. PET imaging and biodistribution data for the two droplet types tested showed significantly different tissue uptake and clearance patterns. CONCLUSION: A convenient method for producing 18F-labeled PFC droplets was developed. The results highlight the potential utility of the strategy for pre-clinical evaluation of different PFC droplet formulations through direct PFC core labeling using a fluorinated radiolabel.

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.

Towards a nanoscale mammographic contrast agent: development of a modular pre-clinical dual optical/x-ray agent.

Contrast-enhanced digital mammography (CEDM) can provide improved breast cancer detection and characterization compared to conventional mammography by imaging the effects of tumour angiogenesis. Current small-molecule contrast agents used for CEDM are limited by a short plasma half-life and rapid extravasation into tissue interstitial space. To address these limitations, nanoscale agents that can remain intravascular except at sites of tumour angiogenesis can be used. For CEDM, this agent must be both biocompatible and strongly attenuate mammographic energy x-rays. Nanoscale perfluorooctylbromide (PFOB) droplets have good x-ray attenuation and have been used in patients for other applications. However, the macroscopic scale of x-ray imaging (50-100 µm) is inadequate for direct verification that PFOB droplets localize at sites of breast tumour angiogenesis. For efficient pre-clinical optimization for CEDM, we integrated an optical marker into PFOB droplets for microscopic assessment (≪50 µm). To develop PFOB droplets as a new nanoscale mammographic contrast agent, PFOB droplets were labelled with fluorescent quantum dots (QDs). The droplets had mean diameters of 160 nm, fluoresced at 635 nm and attenuated x-ray spectra at 30.5 keV mean energy with a relative attenuation of 5.6 ± 0.3 Hounsfield units (HU) mg(-1) mL(-1) QD-PFOB. With the agent loaded into tissue phantoms, good correlation between x-ray attenuation and optical fluorescence was found (R(2) = 0.96), confirming co-localization of the QDs with PFOB for quantitative assessment using x-ray or optical methods. Furthermore, the QDs can be removed from the PFOB agent without affecting its x-ray attenuation or structural properties for expedited translation of optimized PFOB droplet formulations into patients.

Characterization of submicron phase-change perfluorocarbon droplets for extravascular ultrasound imaging of cancer.

Because many tumors possess blood vessels permeable to particles with diameters of 200 nm, it is possible that submicron perfluorocarbon droplets could constitute a novel extravascular ultrasound contrast agent capable of selectively enhancing tumors. Under exposure to bursts of ultrasound of sufficient rarefactional pressure, droplets can undergo vaporization to form echogenic microbubbles. In this study, phase-change thresholds of 220-nm-diameter droplets composed of perfluoropentane were studied in polyacrylamide gel phantoms maintained at temperatures of 21-37°C, exposed to high-pressure bursts of ultrasound with frequencies ranging from 5-15 MHz and durations of 1 µs to 1 ms. The thresholds were found to depend inversely and significantly (p < 0.001) on ultrasound frequency, pulse duration, and droplet temperature, ranging from 9.4 ± 0.8 MPa at 29°C for a 1-µs burst at 5 MHz to 3.2 ± 0.5 MPa at 37°C for a 1-ms burst at 15 MHz. The diameters of microbubbles formed from droplets decreased significantly as phantom stiffness increased (p < 0.0001), and were independent of pulse duration, although substantially more droplets were converted to microbubbles for 1-ms pulse durations compared with briefer exposures. In vivo experiments in a mouse tumor model demonstrated that intravenously injected droplets can be converted into highly echogenic microbubbles 1 h after administration.

Acoustic and photoacoustic characterization of micron-sized perfluorocarbon emulsions.

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.

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.

Single-step coating of mesoporous silica on cetyltrimethyl ammonium bromide-capped nanoparticles.

A generalized, single-step synthesis procedure to coat individual cetyltrimethyl ammonium bromide- (CTAB) capped nanoparticles with a thin layer of mesoporous silica is outlined. This coating method was demonstrated on CTAB-capped Au nanorods and CTAB-transferred CdSe/ZnS quantum dots with silica coatings approximately 15 nm thick containing pores approximately 4 nm in diameter. This porous silica coating can serve as a platform for further surface modification to facilitate the rapid translation of nanoparticles to a wide range of end applications.

Synthesis and optical properties of thiol-stabilized PbS nanocrystals.

Thiol-capped water-soluble PbS nanocrystals (NCs) stabilized with 1-thioglycerol, dithioglycerol, or a mixture of 1-thioglycerol/dithioglycerol (TGL/DTG) were prepared via one-stage synthesis at room temperature. We found that NCs stabilized with a TGL/DTG mixture show efficient and stable infrared photoluminescence centered in the second "biological window" (1050-1200 nm). Under optimized conditions, full width at half-maximum of the PL emission peak was from 70 to 100 nm. PbS NCs were stable to precipitation and aggregation for the time period from 2 to 3 months when stored in the dark under room temperature. Room-temperature photoluminescence quantum efficiency of NCs was from 7 to 10%. When NCs were stored at 37 degrees C, their PL emission red-shifted, consistent with the NC growth.

Hybrid microgels photoresponsive in the near-infrared spectral range.

We report for the first time a photothermally responsive composite material based on polymer microgel particles doped with gold nanorods. We used the dependence of the longitudinal surface plasmon of the gold nanorods on their aspect ratio to synthesize nanoparticles with strong absorption in the near-IR spectral range (in the "water window"). The nanoparticles were incorporated in the interior of temperature-responsive poly(N-isopropylacrylamide-acrylic acid) microgels. Upon irradiation at lambda = 810 nm, hybrid microgel particles doped with Au nanorods underwent a strong deswelling phase transition. These photothermally responsive microgels can be used to carry and release small molecules (e.g., small protein molecules and drugs).