Contrast Agents For Ultrasound Imaging Research Articles

Ultrasound molecular imaging: Moving toward clinical translation

Ultrasound is a widely available, cost-effective, real-time, non-invasive and safe imaging modality widely used in the clinic for anatomical and functional imaging. With the introduction of novel molecularly-targeted ultrasound contrast agents, another dimension of ultrasound has become a reality: diagnosing and monitoring pathological processes at the molecular level. Most commonly used ultrasound molecular imaging contrast agents are micron sized, gas-containing microbubbles functionalized to recognize and attach to molecules expressed on inflamed or angiogenic vascular endothelial cells. There are several potential clinical applications currently being explored including earlier detection, molecular profiling, and monitoring of cancer, as well as visualization of ischemic memory in transient myocardial ischemia, monitoring of disease activity in inflammatory bowel disease, and assessment of arteriosclerosis. Recently, a first clinical grade ultrasound contrast agent (BR55), targeted at a molecule expressed in neoangiogenesis (vascular endothelial growth factor receptor type 2; VEGFR2) has been introduced and safety and feasibility of VEGFR2-targeted ultrasound imaging is being explored in first inhuman clinical trials in various cancer types. This review describes the design of ultrasound molecular imaging contrast agents, imaging techniques, and potential future clinical applications of ultrasound molecular imaging.

Targeted ultrasound contrast agents for ultrasound molecular imaging and therapy

Ultrasound contrast agents (UCAs) are used routinely in the clinic to enhance contrast in ultrasonography. More recently, UCAs have been functionalised by conjugating ligands to their surface to target specific biomarkers of a disease or a disease process. These targeted UCAs (tUCAs) are used for a wide range of pre-clinical applications including diagnosis, monitoring of drug treatment, and therapy. In this review, recent achievements with tUCAs in the field of molecular imaging, evaluation of therapy, drug delivery, and therapeutic applications are discussed. We present the different coating materials and aspects that have to be considered when manufacturing tUCAs. Next to tUCA design and the choice of ligands for specific biomarkers, additional techniques are discussed that are applied to improve binding of the tUCAs to their target and to quantify the strength of this bond. As imaging techniques rely on the specific behaviour of tUCAs in an ultrasound field, it is crucial to understand the characteristics of both free and adhered tUCAs. To image and quantify the adhered tUCAs, the state-of-the-art techniques used for ultrasound molecular imaging and quantification are presented. This review concludes with the potential of tUCAs for drug delivery and therapeutic applications.

Sol-gel synthesis and electrospraying of biodegradable (P2O5)55-(CaO)30-(Na2O)15 glass nanospheres as a transient contrast agent for ultrasound stem cell imaging.

Ultrasound imaging is a powerful tool in medicine because of the millisecond temporal resolution and submillimeter spatial resolution of acoustic imaging. However, the current generation of acoustic contrast agents is primarily limited to vascular targets due to their large size. Nanosize particles have the potential to be used as a contrast agent for ultrasound molecular imaging. Silica-based nanoparticles have shown promise here; however, their slow degradation rate may limit their applications as a contrast agent. Phosphate-based glasses are an attractive alternative with controllable degradation rate and easily metabolized degradation components in the body. In this study, biodegradable P2O5-CaO-Na2O phosphate-based glass nanospheres (PGNs) were synthesized and characterized as contrast agents for ultrasound imaging. The structure of the PGNs was characterized using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), (31)P magic angle spinning nuclear magnetic resonance ((31)P MAS NMR), and Fourier transform infrared (FTIR) spectroscopy. The SEM images indicated a spherical shape with a diameter size range of 200-500 nm. The XRD, (31)P NMR, and FTIR results revealed the amorphous and glassy nature of PGNs that consisted of mainly Q(1) and Q(2) phosphate units. We used this contrast to label mesenchymal stem cells and determined in vitro and in vivo detection limits of 5 and 9 μg/mL, respectively. Cell counts down to 4000 could be measured with ultrasound imaging with no cytoxicity at doses needed for imaging. Importantly, ion-release studies confirmed these PGNs biodegrade into aqueous media with degradation products that can be easily metabolized in the body.

Stimulus-responsive ultrasound contrast agents for clinical imaging: motivations, demonstrations, and future directions.

Microbubble ultrasound contrast agents allow imaging of the vasculature with excellent resolution and signal-to-noise ratios. Contrast in microbubbles derives from their interaction with an ultrasound wave to generate signal at harmonic frequencies of the stimulating pulse; subtracting the elastic echo caused by the surrounding tissue can enhance the specificity of these harmonic signals significantly. The nonlinear acoustic emission is caused by pressure-driven microbubble size fluctuations, which in both theoretical descriptions and empirical measurements was found to depend on the mechanical properties of the shell that encapsulates the microbubble as well as stabilizes it against the surrounding aqueous environment. Thus biochemically induced switching between a rigid 'off' state and a flexible 'on' state provides a mechanism for sensing chemical markers for disease. In our research, we coupled DNA oligonucleotides to a stabilizing lipid monolayer to modulate stiffness of the shell and thereby induce stimulus-responsive behavior. In initial proof-of-principle studies, it was found that signal modulation came primarily from DNA crosslinks preventing the microbubble size oscillations rather than merely damping the signal. Next, these microbubbles were redesigned to include an aptamer sequence in the crosslinking strand, which not only allowed the sensing of the clotting enzyme thrombin but also provided a general strategy for sensing other soluble biomarkers in the bloodstream. Finally, the thrombin-sensitive microbubbles were validated in a rabbit model, presenting the first example of an ultrasound contrast agent that could differentiate between active and inactive clots for the diagnosis of deep venous thrombosis.

Perfluoroalkyl chains as tools for film surface nano-patterning and soft microbubble engineering and decoration

Molecular perfluoroalkylated components provide powerful tools for the elaboration, stabilization and control of organized nano- and micrometre scale soft matter constructs. The examples discussed here include patterning of thin interfacial films with self-assembled organized domains of fluorocarbon/hydrocarbon diblocks, formation and stabilization of size-controlled, resilient soft-shell microbubbles for ultrasound diagnostic imaging, and microbubbles self-assembled from a perfluoroalkylated amphiphile and decorated with iron oxide nanoparticles on their outer surface that are both highly echogenic and magnetic. The latter devices have potential as contrast agents for both ultrasound and magnetic resonance imaging, and as targeted drug delivery agents and devices.

PLGA Hollow Microbubbles Loaded with Iron Oxide Nanoparticles and Doxorubicin for Dual-mode US/MR Imaging and Drug Delivery

We report here the fabrication, characterization and use of PLGA hollow microbubbles (HMs) loaded with iron oxide (Fe3O4) nanoparticles (NPs) and anticancer drug doxorubicin (DOX) for dual-mode ultrasound (US)/magnetic resonance (MR) imaging and drug delivery applications. In this study, a double emulsion technique was employed to prepare PLGA HMs, where hydrophobic superparamagnetic Fe3O4 NPs were encapsulated in the shell of the HMs, while DOX molecules were incorporated within the interior of the PLGA HMs. The formed multifunctional PLGA HMs were characterized via different techniques. We show that the HMs having a size of 1.0 μm are stable and can be potentially used as dual-mode contrast agents for US imaging and MR imaging applications. Furthermore, the HMs encapsulated with DOX are able to release DOX in a sustained manner with a higher release rate under an acidic pH condition than under the physiological pH condition. Importantly, cell viability assay data reveal that the DOX-loaded HMs are able to effectively inhibit the growth of cancer cells with a therapeutic efficacy comparable to free DOX. The fabricated PLGA-Fe3O4-DOX HMs could potentially be used as a theranostic agent for dual-mode US/MR imaging and anticancer drug delivery. Keywords: Doxorubicin, drug delivery, hollow microbubbles, iron oxide nanoparticles, MR imaging, US imaging.

Lipid Shedding from Single Oscillating Microbubbles

Lipid-coated microbubbles are used clinically as contrast agents for ultrasound imaging and are being developed for a variety of therapeutic applications. The lipid encapsulation and shedding of the lipids by acoustic driving of the microbubble has a crucial role in microbubble stability and in ultrasound-triggered drug delivery; however, little is known about the dynamics of lipid shedding under ultrasound excitation. Here we describe a study that optically characterized the lipid shedding behavior of individual microbubbles on a time scale of nanoseconds to microseconds. A single ultrasound burst of 20 to 1000 cycles, with a frequency of 1 MHz and an acoustic pressure varying from 50 to 425 kPa, was applied. In the first step, high-speed fluorescence imaging was performed at 150,000 frames per second to capture the instantaneous dynamics of lipid shedding. Lipid detachment was observed within the first few cycles of ultrasound. Subsequently, the detached lipids were transported by the surrounding flow field, either parallel to the focal plane (in-plane shedding) or in a trajectory perpendicular to the focal plane (out-of-plane shedding). In the second step, the onset of lipid shedding was studied as a function of the acoustic driving parameters, for example, pressure, number of cycles, bubble size and oscillation amplitude. The latter was recorded with an ultrafast framing camera running at 10 million frames per second. A threshold for lipid shedding under ultrasound excitation was found for a relative bubble oscillation amplitude >30%. Lipid shedding was found to be reproducible, indicating that the shedding event can be controlled.

Mechanisms of microbubble-facilitated sonoporation for drug and gene delivery.

Ultrasound-mediated delivery facilitated by microbubbles provides a novel means for intracellular drug and gene delivery and particularly a noninvasive strategy uniquely suitable for clinical applications. Spatiotemporally controllable application of ultrasound energy combined with microbubbles make it possible for site-specific delivery of therapeutic agents to the region-of-interest with minimal undesirable systemic side effects. By inducing rapid expansion/contraction and/or collapse of microbubbles, ultrasound application can temporarily increase the cell membrane permeability (sonoporation) to create a physical route for impermeable agents to enter the cells. Sonoporation is transient and dynamic, involving complex processes of bubble physics, bubble–cell interactions, and subsequent cellular effects that all affect the ultimate delivery outcome. This review summarizes the studies on the important aspects of the mechanisms of ultrasound-mediated delivery, provides illustrative examples of applications, and discusses the challenges and limitations of the technique. Microbubbles have been used for several decades as a contrast agent for ultrasound imaging [1]. The small size of microbubbles allows them access to well-perfused organs when injected into the vasculature, and their gas core efficiently reflects and scatters the incident ultrasound field, thereby increasing image contrast between the vasculature and the surrounding tissue. Recent developments in microbubble technology have enabled molecular imaging via targeting of the microbubbles to molecular markers of disease expressed on the surface of cells [2]. In addition to imaging, innovation in microbubbles has opened new opportunities for targeted drug and gene delivery. Ultrasound excitation of microbubbles has been exploited to increase vascular and cell membrane permeability and facilitate the passage of therapeutic agents across the vascular barrier and cell membrane into the cytoplasm for drug and gene transfection [3–7]. The ultrasound delivery technique, with the advantage of noninvasive, spatiotemporaly controllable ultrasound application combined with functionalized micro-bubbles, holds great promise to provide new therapeutic strategies. However, even with recent progress in the field, challenges remain, including relatively low delivery efficiency and large variation of delivery outcome. Better understanding of the underlying mechanisms is thus of great importance to optimize this technique and promote its translation towards clinical application. Although the mechanisms of ultrasound- and microbubble-facilitated intracellular delivery have not yet been fully understood, a direct physical route of transport often termed sonoporation is most likely involved [8–10]. The dynamic response of the microbubbles driven by ultrasound as well as the interaction between the microbubbles and the cell membrane are key factors in determining delivery efficiency. Other factors such as the cellular response, the kinetics and metabolism of therapeutic agents in the cytoplasm, also play important roles in the ultimate outcome of ultrasound-mediated delivery. In this review, we first summarize progress in these aspects and then discuss limitations and challenges that the technique currently faces.

Preparation of novel curcumin-loaded multifunctional nanodroplets for combining ultrasonic development and targeted chemotherapy

Recently, a new class of multifunctional nanodroplets that combine the properties of polymeric drug carriers, ultrasound imaging contrast agents, and enhancers of ultrasound-mediated drug delivery has been developed. We studied the formation mechanism of nanodroplets of a drug and its application in chemotherapy. Curcumin was loaded in polymeric micelles as a anti-cancer drug using polyethylene glycol block-poly(caprolactone) with encapsulation efficiency of 95.60%. At room temperature, the developed systems comprised perfluorocarbon nanodroplets stabilized by walls comprising biodegradable block copolymers. Upon heating to 37°C, the nanodroplets were converted to nano/microbubbles. Under ultrasound, nanobubbles cavitated and collapsed, resulting in release of the encapsulated drug. The percentage release of curcumin-loaded nanodroplets by insonation was 90.95%, showing enhancement compared with the non-ultrasound group. Nanodroplets strongly retained the loaded drugs in vivo yet, under ultrasound-mediated vaporization, they released the drugs, thereby implementing effective targeting into the tumor. The tumor inhibition of the group in which curcumin-loaded nanodroplets were combined with ultrasound was 71.30%, more than that of the group of curcumin-loaded nanodroplets (53.00%). Nanodroplets showed high enhancement of anti-cancer effects under ultrasound. Upon intravenous injection, a long-lasting, strong and selective ultrasound contrast was observed, suggesting their coalescence into larger, highly echogenic microbubbles. These multifunctional nanodroplets, which manifest excellent therapeutic and ultrasound properties, could be promising anti-cancer drug delivery systems.

Bubble-in-Bubble Strategy for High-Quality Ultrasound Imaging with a Structure Coupling Effect

We design and synthesize monodisperse multi-layer microbubbles (MLBs) with bubble-in-bubble structure using the polymer-backbone-transition method (PBTM). Besides the tunable layer number and microbubble size, we can accurately control the layer-to-layer space and the thickness of each layer. In comparison to traditional single-layer microbubbles, the MLBs can be used as a novel promising model of contrast agent for high-performance ultrasound imaging. The ultrasound signal of MLBs shows a strong structure coupling effect; as the number of layer increases, both the backscatter signal and lifespan increase significantly. We apply a classical acoustic theory to calculate the ultrasound scattering cross-section for the bubbles. It is found that the MLBs exhibit a larger ultrasound scattering cross-section than single-layer bubbles with the same size under certain experimental conditions. Such theoretical results agree well with the ultrasound experimental results.

Methylene blue microbubbles as a model dual-modality contrast agent for ultrasound and activatable photoacoustic imaging

Ultrasound and photoacoustic imaging are highly complementary modalities since both use ultrasonic detection for operation. Increasingly, photoacoustic and ultrasound have been integrated in terms of hardware instrumentation. To generate a broadly accessible dual-modality contrast agent, we generated microbubbles (a standard ultrasound contrast agent) in a solution of methylene blue (a standard photoacoustic dye). This MB2 solution was formed effectively and was optimized as a dual-modality contrast solution. As microbubble concentration increased (with methylene blue concentration constant), photoacoustic signal was attenuated in the MB2 solution. When methylene blue concentration increased (with microbubble concentration held constant), no ultrasonic interference was observed. Using an MB2 solution that strongly attenuated all photoacoustic signal, high powered ultrasound could be used to burst the microbubbles and dramatically enhance photoacoustic contrast (>800-fold increase), providing a new method for spatiotemporal control of photoacoustic signal generation.

Perfluorodecalin nanocapsule as an oxygen carrier and contrast agent for ultrasound imaging

The preparation of a perfluorodecalin (PFD) encapsulated silica nanocapsules via a one-pot synthesis method using Pluronic F68 (PF68) stabilised PFD in water nanoemulsion as a template is demonstrated herein. The method described here simplifies previous multi-step procedures that required pre-fabrication of silica hollow spheres and post-loading of the perfluorocarbon. The size of the PFD nanoemulsion template can be varied easily by changing the concentration of PF68. Successful incorporation of PFD (78% wt) into the silica nanocapsule core and the mechanical stability of the structure were evident from 19F nuclear magnetic resonance analysis; these properties were conferred by the silica-PF68 surface structure. This significant improvement in PFD encapsulation results in higher oxygen carrying capacity compared to encapsulation without PFD as observed from oxygen solubility measurements. The nanocapsules did not show cytotoxicity against HepG2 and 3T3 cells after 24 h exposure at a concentration below 0.8 g L−1 and 0.08 g L−1, respectively. Ultrasound imaging of silica nanocapsules in agarose gel showed a greater than two-fold increment in echogenicity as compared to the PFD nanoemulsion at a similar concentration.

On the interplay of shell structure with low- and high-frequency mechanics of multifunctional magnetic microbubbles

Polymer-shelled magnetic microbubbles have great potential as hybrid contrast agents for ultrasound and magnetic resonance imaging. In this work, we studied US/MRI contrast agents based on air-filled poly(vinyl alcohol)-shelled microbubbles combined with superparamagnetic iron oxide nanoparticles (SPIONs). The SPIONs are integrated either physically or chemically into the polymeric shell of the microbubbles (MBs). As a result, two different designs of a hybrid contrast agent are obtained. With the physical approach, SPIONs are embedded inside the polymeric shell and with the chemical approach SPIONs are covalently linked to the shell surface. The structural design of hybrid probes is important, because it strongly determines the contrast agent's response in the considered imaging methods. In particular, we were interested how structural differences affect the shell's mechanical properties, which play a key role for the MBs' US imaging performance. Therefore, we thoroughly characterized the MBs' geometric features and investigated low-frequency mechanics by using atomic force microscopy (AFM) and high-frequency mechanics by using acoustic tests. Thus, we were able to quantify the impact of the used SPIONs integration method on the shell's elastic modulus, shear modulus and shear viscosity. In summary, the suggested approach contributes to an improved understanding of structure-property relations in US-active hybrid contrast agents and thus provides the basis for their sustainable development and optimization.

The Temperature Rise by Acoustic Radiation Force Impulse under administration of Sonazoid™ (Perfluorobutane): Preliminary Study

The Acoustic Radiation Force Impulse (ARFI) is recently used to evaluate tissue elasticity in some organ. When the biological tissue is exposed by ARFI, the damage by greater ultrasonic pulse and combination with contrast agents is still unknown in both mechanical and thermal effects. Thus, concurrent administration of contrast agent for ultrasound imaging with ARFI had not been advocated, the heat and/or cavitation may occur.

Production of Paclitaxel Loaded PLA-Lecithin Ultrasound Contrast Agents

Drug loaded PLA (or PLGA) mirobubbles that combine properties of ultrasound imaging contrast agents and drug carriers suffer from low encapsulation efficiency and difficulty to destruction with diagnosis ultrasound. In this paper, a new type of multifunctional paclitaxel-loaded poly lactide-lecithin (PLA-lecithin) microbubbles has been developed with a method of ultrasonic double emulsion solvent evaporation (UDES) combined with lyophilization, and single-factor of ultrasonic time was studied to influence bubble size and drug loading efficiency. Bubbles were characterized to be well dispersed, with size of 300nm~2um, and showed increased ultrasound imaging on rabbit liver and heart after intravenous injection.

Epithelial cell biocompatibility of silica nanospheres for contrast-enhanced ultrasound molecular imaging

Nanosized particles are receiving increasing attention as future contrast agents (CAs) for ultrasound (US) molecular imaging, possibly decorated on its surface with biological recognition agents for targeted delivery and deposition of therapeutics. In particular, silica nanospheres (SiNSs) have been demonstrated to be feasible in terms of contrast enhancement on conventional US systems. In this work, we evaluated the cytotoxicity of SiNSs on breast cancer (MCF-7) and HeLa (cervical cancer) cells employing NSs with sizes ranging from 160 to 330 nm and concentration range of 1.5–5 mg/mL. Cell viability was evaluated in terms of size, dose and time dependence, performing the MTT reduction assay with coated and uncoated SiNSs. Whereas uncoated SiNSs caused a variable significant decrease in cell viability on both cell lines mainly depending on size and exposure time, PEGylated SiNSs (SiNSs-PEG) exhibit a high level of biocompatibility. In fact, after 72-h incubation, viability of both cell types was above the cutoff value of 70 % at concentration up to 5 mg/mL. We also investigated the acoustical behavior of coated and uncoated SiNSs within conventional diagnostic US fields in order to determine a suitable configuration, in terms of particle size and concentration, for their employment as targetable CAs. Our results indicate that the employment of SiNSs with diameters around 240 nm assures the most effective contrast enhancement even at the lowest tested concentration, coupled with the possibility of targeting all tumor tissues, being the SiNSs still in a size range where reticuloendothelial system trapping effect is relatively low.

Second Harmonic and Subharmonic for Non-Linear Wideband Contrast Imaging Using a Capacitive Micromachined Ultrasonic Transducer Array

When insonified with suitable ultrasound excitation, contrast microbubbles generate various non-linear scattered components, such as the second harmonic (2H) and the subharmonic (SH). In this study, we exploit the wide frequency bandwidth of capacitive micromachined ultrasonic transducers (CMUTs) to enhance the response from ultrasound contrast agents by selective imaging of both the 2H and SH components simultaneously. To this end, contrast images using the pulse inversion method were recorded with a 64-element CMUT linear array connected to an open scanner. In comparison to imaging at 2H alone, the wideband imaging including both the 2H and SH contributions provided up to 130% and 180% increases in the signal-to-noise and contrast-to-tissue ratios, respectively. The wide-frequency band of CMUTs offers new opportunities for improved ultrasound contrast agent imaging.

Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors

Encapsulated microbubbles are well established as highly effective contrast agents for ultrasound imaging. There remain, however, some significant challenges to fully realize the potential of microbubbles in advanced applications such as perfusion mapping, targeted drug delivery, and gene therapy. A key requirement is accurate characterization of the viscoelastic surface properties of the microbubbles, but methods for independent, nondestructive quantification and mapping of these properties are currently lacking. We present here a strategy for performing these measurements that uses a small fluorophore termed a "molecular rotor" embedded in the microbubble surface, whose fluorescence lifetime is directly related to the viscosity of its surroundings. We apply fluorescence lifetime imaging to show that shell viscosities vary widely across the population of the microbubbles and are influenced by the shell composition and the manufacturing process. We also demonstrate that heterogeneous viscosity distributions exist within individual microbubble shells even with a single surfactant component.

Investigating the effect of fabrication method on the stability and acoustic response of microbubble agents

Microbubbles stabilized by a surfactant or polymer coating are already in clinical use as ultrasound imaging contrast agents. They have also been widely investigated as vehicles for drug delivery and gene therapy that can be tracked and triggered using ultrasound. Extensive studies have been made of the effects of the coating material and gas core on microbubble characteristics, but the influence of the fabrication method has received less attention. The aim of this study was to compare the behavior of microbubbles prepared using different techniques. Phospholipid-coated microbubbles were produced using sonication, electrospraying, or in a specially designed microfluidic device. The microbubbles were observed using optical, electron, and fluorescence lifetime imaging microscopy (FLIM) to interrogate their surface microstructure and stability over time. Their acoustic response was then determined in a flow chamber by detecting the pressure scattered from individual microbubbles as they passed through the focal region of a transducer (center frequencies 1, 2.25, and 3.5 MHz; peak negative pressures 50–300 kPa). The method of bubble generation was found to significantly affect the bubble surface characteristics, stability, and acoustic response. The results demonstrate that the processing method affects not only the bubble size distribution but other characteristics important for biomedical applications.

Cyanine 5.5 conjugated nanobubbles as a tumor selective contrast agent for dual ultrasound-fluorescence imaging in a mouse model.

Nanobubbles and microbubbles are non-invasive ultrasound imaging contrast agents that may potentially enhance diagnosis of tumors. However, to date, both nanobubbles and microbubbles display poor in vivo tumor-selectivity over non-targeted organs such as liver. We report here cyanine 5.5 conjugated nanobubbles (cy5.5-nanobubbles) of a biocompatible chitosan–vitamin C lipid system as a dual ultrasound-fluorescence contrast agent that achieved tumor-selective imaging in a mouse tumor model. Cy5.5-nanobubble suspension contained single bubble spheres and clusters of bubble spheres with the size ranging between 400–800 nm. In the in vivo mouse study, enhancement of ultrasound signals at tumor site was found to persist over 2 h while tumor-selective fluorescence emission was persistently observed over 24 h with intravenous injection of cy5.5-nanobubbles. In vitro cell study indicated that cy5.5-flurescence dye was able to accumulate in cancer cells due to the unique conjugated nanobubble structure. Further in vivo fluorescence study suggested that cy5.5-nanobubbles were mainly located at tumor site and in the bladder of mice. Subsequent analysis confirmed that accumulation of high fluorescence was present at the intact subcutaneous tumor site and in isolated tumor tissue but not in liver tissue post intravenous injection of cy5.5-nanobubbles. All these results led to the conclusion that cy5.5-nanobubbles with unique crosslinked chitosan–vitamin C lipid system have achieved tumor-selective imaging in vivo.