Nanoscale Ultrasound Contrast Agents Research Articles

Nonlinear Frequency Mixing Ultrasound Imaging of Nanoscale Contrast Agents.

Nanoscale ultrasound contrast agents show promise as alternatives for diagnostics and therapies due to their enhanced stability and ability to traverse blood vessels. Nonetheless, their reduced size limits echogenicity. This study introduces an enhanced nanobubble frequency mixing ultrasound imaging method, by capitalizing on their nonlinear acoustic response to dual-frequency excitation. A single broadband transducer (L12-3v) controlled by a programmable ultrasound system was used to transmit a dual-frequency single-cycle wavefront. The frequency mixing effect enabled simultaneous transducer capture of nanobubble-generated sum and difference frequencies in real time without the need for additional hardware or post-processing, by substituting the single-frequency wavefront in a standard contrast harmonic pulse inversion imaging protocol, with the dual-frequency wavefront. Optimization experiments were conducted in tissue mimicking phantoms. Among the dual-frequency combinations that were tested, the highest contrast was obtained using 4&8 MHz. The nanobubble contrast improved with increased mechanical index, and achieved a maximal contrast improvement of 8.4 ± 0.5 dB compared to 4 MHz pulse inversion imaging. In imaging of a breast cancer tumor mouse model, after a systemic nanobubble injection, the contrast was improved by 3.4 ± 1.7, 4.8 ± 1.8, and 6.3 ± 1.6 dB for mechanical indices of 0.04, 0.08, and 0.1, respectively. Nonlinear frequency mixing significantly improved the nanobubble contrast, which facilitated their imaging in-vivo. This study offers a new avenue to enhance ultrasound imaging utilizing nanobubbles, potentially leading to advancements in other diagnostic applications.

Sono-Immunotherapy Mediated Controllable Composite Nano Fluorescent Probes Reprogram the Immune Microenvironment of Hepatocellular Carcinoma.

Despite the clinical efficacy of immunotherapy in treating malignant tumors, its effectiveness is often hampered by the immunosuppressive nature of the tumor microenvironment (TME). In this study, we propose the design of a nanoscale ultrasound contrast agent capable of triggering macrophage polarization and immunogenic cell death (ICD) for the treatment of hepatocellular carcinoma (HCC) through sonodynamic treatment (SDT) and immunotherapy. The re-educator (designated as ICG@C3F8-R848 NBs) is composed of the Toll-like receptor agonist resiquimod (R848) and the sonosensitizer Indocyanine green (ICG), utilizing nanobubbles (NBs) as carriers. The technique known as ultrasound-targeted nanobubble destruction (UTND) employs nanosized microbubbles and low-frequency ultrasound (LFUS) to ensure accurate drug delivery and enhance safety. Following intravenous delivery, ICG@C3F8-R848 NBs have the potential to selectively target and treat primary tumors using SDT in conjunction with ultrasonography. Importantly, R848 can enhance antitumor immunity by inducing the polarization of macrophages from an M2 to an M1 phenotype. The SDT-initiated immunotherapy utilizing ICG@C3F8-R848 NBs demonstrates significant tumor suppression effects with minimal risk of systemic toxicity. The utilization of this self-delivery re-education technique would contribute to advancing the development of nanomedicine for the treatment of hepatocellular carcinoma.

Ultrasound targeted microbubble destruction-triggered nitric oxide release via nanoscale ultrasound contrast agent for sensitizing chemoimmunotherapy

Immunotherapy had demonstrated inspiring effects in tumor treatment, but only a minority of people could benefit owing to the hypoxic and immune-suppressed tumor microenvironment (TME). Therefore, there was an urgent need for a strategy that could relieve hypoxia and increase infiltration of tumor lymphocytes simultaneously. In this study, a novel acidity-responsive nanoscale ultrasound contrast agent (L-Arg@PTX nanodroplets) was constructed to co-deliver paclitaxel (PTX) and L-arginine (L-Arg) using the homogenization/emulsification method. The L-Arg@PTX nanodroplets with uniform size of about 300 nm and high drug loading efficiency displayed good ultrasound diagnostic imaging capability, improved tumor aggregation and achieved ultrasound-triggered drug release, which could prevent the premature leakage of drugs and thus improve biosafety. More critically, L-Arg@PTX nanodroplets in combination with ultrasound targeted microbubble destruction (UTMD) could increase cellular reactive oxygen species (ROS), which exerted an oxidizing effect that converted L-Arg into nitric oxide (NO), thus alleviating hypoxia, sensitizing chemotherapy and increasing the CD8 + cytotoxic T lymphocytes (CTLs) infiltration. Combined with the chemotherapeutic drug PTX-induced immunogenic cell death (ICD), this promising strategy could enhance immunotherapy synergistically and realize powerful tumor treatment effect. Taken together, L-Arg@PTX nanodroplets was a very hopeful vehicle that integrated drug delivery, diagnostic imaging, and chemoimmunotherapy.Graphical

Role of Surface Charge of Nanoscale Ultrasound Contrast Agents in Complement Activation and Phagocytosis.

To prepare nanoscale ultrasound contrast agents (Nano-UCAs) and examine the role of their surface charge in complement activation and phagocytosis. We analyzed serum proteins present in the corona formed on Nano-UCAs and evaluated two important protein markers of complement activation (C3 and SC5b-9). The effect of surface charge on phagocytosis was further assessed using THP-1 macrophages. When Nano-UCAs were incubated with human serum, they were opsonized by various blood proteins, especially C3. Highly charged Nano-UCAs, whether positive or negative, were favorably opsonized by complement proteins and phagocytized by macrophages. Charged Nano-UCAs show a higher tendency to activated complement system, and are efficiently engulfed by macrophages. The present results provide meaningful insights into the role of the surface charge of nanoparticles in the activation of the innate immune system, which is important not only for the design of targeted Nano-UCAs, but also for the effectiveness and safety of other theranostic agents.

A New Nanoscale Ultrasound Phase-Variant Contrast Agent for Phase Variant Low-Frequency Medical Ultrasound Imaging That Can Scavenge Reactive Oxygen Species

Nanoscale phase-variant ultrasound contrast agents have attracted the interest of many researchers. However, it is a challenge to design nanobubbles that are activated by low-frequency medical ultrasound that do not cause damage to normal tissues. In this study, we designed a new type of nanoparticle consisting of perfluoropentane and iron polyphthalocyanine loaded into liposomes. These nanoparticles can be activated by a low-frequency medical ultrasound imager at a frequency of 5 MHz for ultrasound imaging and clear reactive oxygen species at a rate of more than 50%. This ability to scavenge excess reactive oxygen species can alleviate the damage these species cause and protect macrophages. Moreover, these nanoparticles can effectively enhance ultrasound contrast imaging for real-time visualization in the diagnosis and treatment of diseases.

Nanoscale Contrast Agents for Ultrasound Imaging of Musculoskeletal System.

Musculoskeletal ultrasound (MSKUS) has been recognized as an important method for the evaluation of diseases of the musculoskeletal system, and contrast-enhanced ultrasound (CEUS) technology is becoming an important branch of it. The development of novel materials and tiny nano-formulations has further expanded ultrasound contrast agents (UCAs) into the field of nanotechnology. Over the years, nanoscale contrast agents have been found to play an unexpected role in the integration of precise imaging for diagnosis and treatment of numerous diseases. It has been demonstrated that nanoscale UCAs (nUCAs) have advantages in imaging over conventional contrast agents, including superior biocompatibility, serum stability, and longer lifetime. The potential value of nUCAs in the musculoskeletal system is that they provide more reliable and clinically valuable guidance for the diagnosis, treatment, and follow-up of related diseases. The frontier of advances in nUCAs, their applications, and insights in MSKUS are reviewed in this paper.

Characterization of the interaction of nanobubble ultrasound contrast agents with human blood components

Nanoscale ultrasound contrast agents, or nanobubbles, are being explored in preclinical applications ranging from vascular and cardiac imaging to targeted drug delivery in cancer. These sub-micron particles are approximately 10x smaller than clinically available microbubbles. This allows them to effectively traverse compromised physiological barriers and circulate for extended periods of time. While various aspects of nanobubble behavior have been previously examined, their behavior in human whole blood has not yet been explored. Accordingly, herein we examined, for the first time, the short and long-term effects of blood components on nanobubble acoustic response. We observed differences in the kinetics of backscatter from nanobubble suspensions in whole blood compared to bubbles in phosphate buffered saline (PBS), plasma, or red blood cell solutions (RBCs). Specifically, after introducing nanobubbles to fresh human whole blood, signal enhancement, or the magnitude of nonlinear ultrasound signal, gradually increased by 22.8 ± 13.1% throughout our experiment, with peak intensity reached within 145 s. In contrast, nanobubbles in PBS had a stable signal with negligible change in intensity (−1.7 ± 3.2%) over 8 min. Under the same conditions, microbubbles made with the same lipid formulation showed a −56.8 ± 6.1% decrease in enhancement in whole blood. Subsequent confocal, fluorescent, and scanning electron microscopy analysis revealed attachment of the nanobubbles to the surface of RBCs, suggesting that direct interactions, or hitchhiking, of nanobubbles on RBCs in the presence of plasma may be a possible mechanism for the observed effects. This phenomenon could be key to extending nanobubble circulation time and has broad implications in drug delivery, where RBC interaction with nanoparticles could be exploited to improve delivery efficiency.

Detection of Cardiac Functions of Fetus with Diabetic Metabolic Disease through PEG-PCLNano Micelle and Ultrasound Technique

210 patients with a singleton pregnancy fetus, who received the fetal echocardiography in Yuhang District First People's Hospital from March 2019 to March 2020, were selected as the subjects, including 101 fetuses who were confirmed as gestational diabetes mellitus(GDM), and 109 normal fetuses (control group). The ultrasound cardiogram technique was employed to detect the thickness of the fetus ventricle septum, mitral/tricuspid annular displacement, left/right TEI indexes, and so on. The mean value of three cardiac cycles was taken as the test results. Finally, SPSS17.0 software was applied to the analysis of data. The nano micelle was made from the amphiphilic block copolymers (PEG-PCL) using the dialysis method/solvent evaporation method. The nanoscale ultrasound contrast agent was prepared from Decafluoropentane which was imaging gas. The characterizations were studied using the optical microscope, and transmission electron microscopy (TEM). The temperature sensitivity and ultrasound sensitivity of the nano-ultrasound contrast agent were analyzed with the particle size as the evaluation index. The in-vitro ultrasound contrast experiment was conducted to study the contrast-enhanced effect. The fetal Tei index of the case group was higher than that of the control group, of which P<0.05 had statistical significance. However, the thickness of the fetus ventricle septum, Em, Am, and Em/Am of mitral/tricuspid annular were not significantly different from those of the control group (P>0.05). The nano ultrasonic contrast agent prepared through the ultrasonic injection method had a uniform particle size and a hollow shell-core structure under an electron projection microscope. The particle size of the nano-ultrasound contrast agent varied with temperature, and its microbubbles were generated under ultrasonic conditions. As compared with the blank degassed water group, a real linear echo appeared inside the contrast agent group, with small and even echo spots. The back echo remained with no obvious attenuation and lasted for a longer period. However, the blank degassed group had no distinct echo intensity and spot. PEG-PCL nano-ultrasound contrast agent achieved an excellent imaging effect; there was no obvious change to heart function and structure of the fetus, when gestational diabetes pregnant had blood sugar perfectly controlled, however, the fetus's heart function may change in the last trimester.

Effects of shell-integrated Sudan Black dye on the acoustic activity and ultrasound imaging properties of lipid-shelled nanoscale ultrasound contrast agents.

.Significance: An effective contrast agent for concurrent multimodal photoacoustic (PA) and ultrasound (US) imaging must have both high optical absorption and high echogenicity. Integrating a highly absorbing dye into the lipid shell of gas core nanobubbles (NBs) adds PA contrast to existing US contrast agents but may impact agent ultrasonic response.Aim: We report on the development and ultrasonic characterization of lipid-shell stabilized NBs with integrated Sudan Black (SB) B dye in the shell as dual-modal PA-US contrast agents.Approach: Perfluoropropane NBs stabilized with a lipid shell including increasing concentrations of SB B dye were formulated by amalgamation (SBNBs). Physical properties of SBNBs were characterized using resonant mass measurement, transmission electron microscopy and pendant drop tensiometry. Concentrated bubble solutions were imaged for 8 min to assess signal decay. Diluted bubble solutions were stimulated by a focused transducer to determine the response of individual NBs to long cycle (30 cycle) US. For assessment of simultaneous multimodal contrast, bulk populations of SBNBs were imaged using a PA and US imaging platform.Results: We produced high agent yield () with a mean diameter of to 300 nm depending on SB loading. A 40% decrease in bubble yield was measured for solutions with 0.3 and SB. The addition of SB to the shell did not substantially affect NB size despite an increase in surface tension by up to . The bubble decay rate increased after prolonged exposure (8 min) by dyed bubbles in comparison to their undyed counterparts (2.5-fold). SB in bubble shells increased gas exchange across the shell for long cycle US. PA imaging of these agents showed an increase in power (up to 10 dB) with increasing dye.Conclusions: We added PA contrast function to NBs. The addition of SB increased gas exchange across the NB shell. This has important implications in their use as multimodal agents.

Molecular imaging diagnosis of atherosclerotic vulnerable plaque in rabbit carotid artery using a self-assembled nanoscale ultrasound microbubble contrast agent.

This study aimed to prepare an anti-Vascular cell adhesion protein 1 (VCAM-1) nanoscale ultrasound microbubble contrast agent using the hyperbranched self-assembly method for the molecular imaging diagnosis of atherosclerotic vulnerable plaques in rabbits. Twenty-five rabbits with carotid atherosclerosis were randomly divided into 5 groups, and the ear vein was injected with agents as follows: Groups A and B: nanoscale ultrasound microbubble contrast agent with and without anti-VCAM-1 agent; Groups C and D: SonoVue ultrasonic microbubble contrast agent, with and without anti-VCAM-1 agent; Control group: saline. The molecular imaging diagnosis of the atherosclerotic plaque, involved the examination of its vulnerability in the rabbit carotid artery was performed using the contrast ultrasound mode. The arrival and peaking time of the anti-VCAM-1 nanoscale ultrasound microbubble contrast agent (Group A) for plaque occurred earlier than those of the other groups (p < 0.05), and with it, the plaque showed the strongest enhancement (p < 0.05), followed by the SonoVue ultrasound microbubble contrast agent with anti-VCAM-1 group (Group C) and the self-made nanoscale ultrasound microbubble contrast agent group (Group B). No development was observed in the plaques of the SonoVue ultrasound microbubble contrast agent group and the control group. The anti-VCAM-1 nanoscale ultrasonic microbubble contrast agent, prepared using the self-assembly method, can facilitate the development effect of the carotid atherosclerotic vulnerable plaque, providing a basis for the molecular imaging diagnosis of carotid atherosclerotic vulnerable plaques.

Novel biomimetic dual-mode nanodroplets as ultrasound contrast agents with potential ability of precise detection and photothermal ablation of tumors.

Molecule-targeted ultrasound imaging has attracted extensive attention for precise diagnosis and targeted therapy of tumors. The aim of this research is to prepare novel biomimetic dual-mode nanoscale ultrasound contrast agents (UCAs), which can not only evade the immune clearance of reticuloendothelial system, but also have the potential ability of precise detection and photothermal ablation of tumors. In this study, for the first time, the novel biomimetic UCAs were prepared by encapsulating liquid perfluorohexanes with red blood cell membranes carrying IR-780 iodide and named IR780-RBCM@NDs. The characteristics of that were verified through the particle size analyzer, scanning electron microscopy, transmission electron microscopy and laser scanning confocal microscopy. The stability of IR780-RBCM@NDs at 37°C was explored. The abilities of immune escape, dual-mode imaging and photothermal effect for IR780-RBCM@NDs were verified via in vitro experiments. The novel prepared nanodroplets have good characteristics such as mean diameter, zeta potential, and relatively stability. Importantly, the integrin-associated protein expressed on the surface of RBCMs was detected on IR780-RBCM@NDs. Then, compared with control groups, IR780-RBCM@NDs performed excellent immune escape function away from macrophages in vitro. Furthermore, the IR-780 iodide was observed on the new nanodroplets and that was able to perform the dual-mode imaging with near-infrared fluorescence imaging and contrast-enhanced ultrasound imaging after the phase change. Finally, the effective photothermal ablation ability of IR780-RBCM@NDs was verified in tumor cells. The newly prepared biomimetic IR780-RBCM@NDs provided novel ideas for evading immune clearance, performing precise diagnosis and photothermal ablation of tumor cells.

Preparation of Lipid Nano-Bubbles as Contrast Agent Material for Tumor Ultrasound Diagnosis of Breast Cancer

Ultrasound diagnostic technology has higher application values in tumor diagnosis due to its higher spatial resolution and no radiation. Ultrasound lipid nanobubble contrast material can further improve the accuracy of tumor diagnosis. Therefore, the liposomes were used as shells to encapsulate bio-inert gases to prepare nano-scale microbubbles NBs. Meanwhile, the Annexin V molecules were connected through the conjugation of biotin-avidin to prepare targeted nano-scale ultrasound contrast agents (UCAs) Annexin V-Nanobubbles (AVNBs). The tumor-bearing mice apoptosis models were constructed by injecting human breast cancer MDA-MB231 cells, and the nano-scale UCA AV-NBs was used for ultrasound diagnosis, data collection, and statistical analysis. The results showed that the prepared AV-NBs were nano-sized hollow spheres with relatively stable particle size, zeta potential, and excellent stability. By verifying the constructed mice apoptosis models, it was found that mice heterotopic transplantation tumor apoptosis models were successfully constructed. When the mice models were observed by ultrasound, the apoptosis index of the cell was found to be positively correlated with the enhancement degree of the image. Therefore, the enhancement effect of the targeted UCA AV-NBs was obvious. The AV-NBs could be specifically targeted to the apoptotic region of tumor tissue in vivo, which was also slightly enriched in the liver and kidney. Therefore, the experiment found that the targeted nano-scale UCA AV-NBs combined with ultrasound detection could significantly enhance the diagnostic accuracy of breast cancer tumors, which provided the experimental evidence for early tumor diagnosis and evaluation.

Site-targeted imaging enhancement of viable myocardium after ischemia-reperfusion by a novel nano-scale ultrasound contrast agent: a vivo study

Objective To prepare a kind of lipid nanoparticle ultrasound contrast agents with the ability to target to viable myocardium for diagnosis. Methods The agent was a biotinylated, fluorescent-labelled, lipid-coated, liquid perfluorocarbon emulsion. Physico-chemical properties of the agent were measured, including size distribution, Zeta Potential, concentration and so on. Ischemia-reperfusion models were created in rats, and then exposed to biotinylated anti-MCP-1 monoclonal antibody, rhodamine avidin and biotinylated, FITC-labelled nanoparticles, respectively. Echocardiography was taken before and after injection. Frozen sections of their hearts were observed under fluorescence microscope. Results The particle diameter, zeta potential and concentration of lipid nanoparticles were (172.30±52.06)nm, (-33.10±6.50)mV and (2.28±0.46)×1011/ml, respectively. From the short-axis view, the myocardium under endocardium of anterior wall was enhanced obviously. While myocardium of other walls were still. The lipid nanoparticles located in the myocardium of anterior wall and gave out bright green and red fluorescence under fluorescence microscope, while neither lipid nanoparticles nor fluorescence were found in other sites of ventricular myocardium. Conclusions The viable myocardium can be targeted and acoustically enhanced by the self-made nano-scale ultrasound contrast agent. This new agent has potential to improve sensitivity and specificity for noninvasive identifying viable myocardium. Key words: Ultrasound contrast agents; Nano-scale; Perfluorocarbon; Viable myocardium

Indocyanine green (ICG) – Menthol loaded cerasomal nanoparticles for ultrasound imaging and photothermal therapy against tumor

Abstract Multifunctional ultrasound contrast agents (UCAs) combining ultrasonic diagnosis with tumor therapy have received enormous attentions in virtue of their good biocompatibility and high biological penetration ability. However, nanoscale UCAs with suitable size and good therapeutic effect remain to be developed in future studies. Herein, indocyanine green and L-menthol loaded hybrid cerasomes (ICG-MCNs) was fabricated as a multifunctional theranostic nanoplatform, which exhibited high structure stability and photo-stability in aqueous. Mutual assistance with the photothermal effect of ICG, heating triggered tri-phase transition of L-menthol could produce continuous gas microbubbles for enhanced ultrasound imaging. Thus, the resultant ICG-MCNs is anticipated to be a competent candidate for US imaging and photothermal therapy.

Effect of Bubble Concentration on the in Vitro and in Vivo Performance of Highly Stable Lipid Shell-Stabilized Micro- and Nanoscale Ultrasound Contrast Agents.

Ultrasound (US) is a widely used diagnostic imaging tool because it is inexpensive, safe, portable, and broadly accessible. Ultrasound contrast agents (UCAs) are employed to enhance backscatter echo and improve imaging contrast. The most frequently utilized UCAs are echogenic bubbles made with a phospholipid or protein-stabilized hydrophobic gas core. While clinically utilized, applications of UCAs are often limited by rapid signal decay (<5 min) in vivo under typical ultrasound imaging protocols. Here, we report on a formulation of lipid shell-stabilized perfluoropropane (C3F8) microbubbles and nanobubbles with a significantly prolonged in vivo stability. Microbubbles (875 ± 280 nm) of the target size were prepared by utilizing a multiple-step centrifugation cycle, while nanobubbles (299 ± 189 nm) were isolated from the activated vial using a single centrifugation step. To provide in-depth acoustic characterization of the new construct we evaluated the effect of size and concentration on their in vitro and in vivo performance. In vitro and in vivo characterization were carried out for a range of bubble concentrations normalized by total gas volume quantified via headspace gas chromatography/mass spectrometry (GC/MS). In vitro characterization revealed that nanobubbles at different concentrations are more consistently stable over time with the highest and lowest dilutions (50-fold decrease) only differing in US signal after 8 min exposure by 10.34%, while for microbubbles the difference was 86.46%. As expected, due to the difference in hydrodynamic diameter and scattering cross section difference, nanobubbles showed lower overall initial signal intensity. In vivo experiments showed that both microbubbles and nanobubbles with similar initial peak signal intensity are comparably stable over time with 66.8% and 60.6% remaining signal after 30 min, respectively. This study demonstrates that bubble concentration has significant effects on the persistence of both microbubbles and nanobubbles in vitro and in vivo, but the effects are more pronounced in larger bubbles. These effects should be taken into account when selecting the appropriate bubble parameters for future imaging applications.

Ultrasound Technology for Molecular Imaging: From Contrast Agents to Multimodal Imaging.

Ultrasound (US) takes advantage of ultrasound contrast agents (UCAs) to further increase the sensitivity and specificity of monitoring at the cellular level, which has had a considerable effect on the modern molecular imaging field. Gas-filled microbubbles (MBs) as UCAs in the bloodstream generate resonant volumetric oscillations in response to rapid variations in acoustic pressure, which are related to both the acoustic parameters of applied ultrasound and the physicochemical properties of the contrast agents. Nanoscale UCAs have been developed and have attracted much attention due to their utility in detecting extravascular lesions. Ultrasound molecular assessment is achieved by binding disease-specific ligands to the surface of UCAs, which have been designed to target tissue biomarkers in the area of interest, such as blood vessels, inflammation, or thrombosis. Additionally, the development of multimodal imaging technology is conducive for integration of the advantages of various imaging techniques to acquire additional diagnostic information. In this review paper, the present status and the critical issues for developing ultrasound contrast agents and multimodal imaging applications are described. Conventional MB UCAs are first introduced, including their research material, diagnostic applications, and intrinsic limitations. Then, recent progress in developing targeted UCAs and phase-inversion contrast agents for diagnostic purposes is discussed. Finally, we review the present status and the critical issues for developing ultrasound-based multimodal imaging applications and summarize the existing challenges and future prospects.

Preparation and characterization of a novel silicon-modified nanobubble.

Nanobubbles (NBs) opened a new field of ultrasound imaging. There is still no practical method to control the diameter of bubbles. In this study, we developed a new method to control the size by incorporating of silicon hybrid lipids into the bubble membrane. The range of particle size of resulting NBs is between 523.02 ± 46.45 to 857.18 ± 82.90, smaller than the conventional microbubbles. The size of resulting NBs increased with the decrease in amount of silicon hybrid lipids, indicating the diameter of NBs can be regulated through modulating the ratio of silicon hybrid lipids in the bubble shell. Typical harmonic signals could be detected. The in vitro and in vivo ultrasound imaging experiments demonstrated these silicon-modified NBs had significantly improved ultrasound contrast enhancement abilities. Cytotoxicity assays revealed that these NBs had no obvious cytotoxicity to the 293 cell line at the tested bubble concentration. Our results showed that the novel NBs could use as nanoscale ultrasound contrast agents, providing the foundation for NBs in future applications including contrast-enhanced imaging and drug/gene delivery.

Recent advances in ultrasound-based diagnosis and therapy with micro- and nanometer-sized formulations.

Ultrasound (US) is one of the most frequently used imaging methods in the clinic. The broad spectrum of its applications can be increased by the use of gas-filled microbubbles (MB) as ultrasound contrast agents (UCA). In recent years, also nanoscale UCA like nanobubbles (NB), echogenic liposomes (ELIP) and nanodroplets have been developed, which in contrast to MB, are able to extravasate from the vessels into the tissue. New disease-specific UCA have been designed for the assessment of tissue biomarkers and advanced US to a molecular imaging modality. For this purpose, specific binding moieties were coupled to the UCA surface. The vascular endothelial growth factor receptor-2 (VEGFR-2) and P-/E-selectin are prominent examples of molecular US targets to visualize tumor blood vessels and inflammatory diseases, respectively. Besides their application in contrast-enhanced imaging, MB can also be employed for drug delivery to tumors and across the blood-brain barrier (BBB). This review summarizes the development of micro- and nanoscaled UCA and highlights recent advances in diagnostic and therapeutic applications, which are ready for translation into the clinic.

Superhydrophobic silica nanoparticles as ultrasound contrast agents

Microbubbles have been widely studied as ultrasound contrast agents for diagnosis and as drug/gene carriers for therapy. However, their size and stability (lifetime of 5–12min) limited their applications. The development of stable nanoscale ultrasound contrast agents would therefore benefit both. Generating bubbles persistently in situ would be one of the promising solutions to the problem of short lifetime. We hypothesized that bubbles could be generated in situ by providing stable air nuclei since it has been found that the interfacial nanobubbles on a hydrophobic surface have a much longer lifetime (orders of days). Mesoporous silica nanoparticles (MSNs) with large surface areas and different levels of hydrophobicity were prepared to test our hypothesis. It is clear that the superhydrophobic and porous nanoparticles exhibited a significant and strong contrast intensity compared with other nanoparticles. The bubbles generated from superhydrophobic nanoparticles sustained for at least 30min at a MI of 1.0, while lipid microbubble lasted for about 5min at the same settings. In summary MSNs have been transformed into reliable bubble precursors by making simple superhydrophobic modification, and made into a promising contrast agent with the potentials to serve as theranostic agents that are sensitive to ultrasound stimulation.

Biodegradable double-targeted PTX-mPEG-PLGA nanoparticles for ultrasound contrast enhanced imaging and antitumor therapy in vitro.

A porous-structure nano-scale ultrasound contrast agent (UCA) was made of monomethoxypoly (ethylene glycol)-poly (lactic-co-glycolic acid) (mPEG-PLGA), and modified by double-targeted antibody: anti-carcinoembryonic antigen (CEA) and anti-carbohydrate antigen 19-9 (CA19-9), as a double-targeted nanoparticles (NPs). Anti-tumor drug paclitaxel (PTX) was encapsulated in the double-targeted nanoparticles (NPs). The morphor and release curve were characterized. We verified a certain anticancer effect of PTX-NPs through cytotoxicity experiments. The cell uptake result showed much more NPs may be facilitated to ingress the cells or tissues with ultrasound (US) or ultrasound targeted microbubble destruction (UTMD) transient sonoporation in vitro. Ultrasound contrast-enhanced images in vitro and in vivo were investigated. Compared with SonoVue, the NPs prolonged imaging time in rabbit kidneys and tumor of nude mice, which make it possible to further enhance anti-tumor effects by extending retention time in the tumor region. The novel double-targeted NPs with the function of ultrasound contrast enhanced imaging and anti-tumor therapy can be a promising way in clinic.