Nanobubble Contrast Agent Research Articles

Superresolution imaging of microvasculature at the capillary level using nanobubble contrast agents

In this work, we describe applications of nanobubbles in superresolution imaging. Conventional ultrasound localization microscopy (ULM) techniques require sparse scatterers in the form of echogenic microbubbles (MB, ∼1–10 μm diameter). ULM enables imaging of capillaries larger than ∼50 μm in diameter but becomes too time consuming in smaller capillaries due to insufficient MB density. A viable alternative for microvascular imaging may be the use of submicron bubbles, or nanobubbles (NBs). NBs are ∼100i–500 nm in diameter and have a particle density 5 orders of magnitude higher than MBs without associated attenuation artifacts. In addition to strong nonlinear response, under some conditions NBs can produce SNR linear signals stronger than erythrocytes. This allows for formulating a superresolution imaging problem using computational approaches suitable for dense sources instead of relying on physical sparsity. With much smaller sizes compared to MBs, NBs can access the smallest capillaries at much higher concentrations (∼1011), offering enhanced detection and faster superresolution acquisition. In addition, NB nonlinear signals can be decomposed to elucidate intravascular and extravasation into tissues with vascular hyperpermeability, such as tumors. Thus, when combined with appropriate processing, NBs can provide information about vascular architecture and extravascular transport which is not achievable with MBs.

Endoleak Detection after Endovascular Aortic Aneurysm Repair Using Ultrasound Based on Nanoscale Bubble Contrast Agents and Their Effects on Vascular Smooth Muscle Cell Proliferation and Migration

Abdominal aortic aneurysm (AAA) is the most common vascular disease that causes disability and death. Its morbidity is relatively subtle, and the mortality rate is high. Clinically, endovascular aortic aneurysm repair (EVAR) has gradually become the primary treatment of AAA due to its unique advantages such as low trauma and low incidence of short-term complications. However, the outcome of EVAR is greatly compromised by the possible occurrence of endoleaks. Contrast-enhanced ultrasound (CEUS) is a promising alternative technique to detect endoleaks following EVAR due to lack of exposure to ionizing radiation. Traditional ultrasound contrast agents with an overlarge size (microscale) leading to reluctant accumulation in target organs and instability trigger the requirement of nanoscale contrast agents that enter tumor tissues through the enhanced permeability and retention effect. In this study, we used ultrasound based on nanoscale bubble contrast agents to evaluate endoleak detection after endovascular aortic aneurysm repair and analyzed the effects of nanoscale bubble contrast agents on vascular smooth muscle cell (VSMC) proliferation and migration. Among 52 AAA patients a month following EVAR, there were 16 cases of endoleaks after EVAR detected by nanobubble contrast-enhanced ultrasound, including 6 cases of type I endoleak (1 case of type Ia endoleak and 5 cases of type Ib endoleak), 7 cases of type II endoleak, and 3 cases of type III endoleak; there were 12 cases of endoleak after EVAR detected by computed tomography angiography (CTA), including 6 cases of type I endoleak (1 case of type Ia and 5 cases of type Ib), 5 cases of type II endoleak, and 1 case of type III endoleak. Six months after EVAR, 3 cases of type III endoleak were detected by both nanobubble contrast-enhanced ultrasound and CTA. Vascular smooth muscle cells (VSMCs) used for in vitro experiments were subjected to ultrasound irradiation and platelet-derived growth factor (PDGF) treatment with or without the addition of nanobubble contrast agents. After high-intensity and long-term irradiation (0.75 W/cm2 and 1 W/cm2 irradiation for 120 s and 150 s, respectively) by ultrasound with or without the addition of nanobubble contrast agents, PDGF-induced VSMC migration was inhibited ( P < 0.01 ). Low-intensity and short-term ultrasound irradiation did not differ PDGF-induced VSMC migration ( P > 0.05 ), but 0.5 W/cm2 and 90 s ultrasound irradiation could significantly inhibit PDGF-induced VSMC migration without the addition of nanobubble contrast agents ( P < 0.05 ). When VSMCs were irradiated at 1, 0.75, 0.5, and 0.35 W/cm2 for 30 s to 150 s, ultrasound irradiation with or without the addition of nanobubble contrast agents remarkably reduced PDGF-induced VSMC proliferation, as evidenced by reduced OD values ( P < 0.05 ). In conclusion, ultrasound based on nanoscale bubble contrast agents is an effective alternative detection method for the occurrence of AAA patients who are not suitable for CTA.

Ultrasound-Based Molecular Imaging of Tumors with PTPmu Biomarker-Targeted Nanobubble Contrast Agents.

Ultrasound imaging is a widely used, readily accessible and safe imaging modality. Molecularly-targeted microbubble- and nanobubble-based contrast agents used in conjunction with ultrasound imaging expand the utility of this modality by specifically targeting and detecting biomarkers associated with different pathologies including cancer. In this study, nanobubbles directed to a cancer biomarker derived from the Receptor Protein Tyrosine Phosphatase mu, PTPmu, were evaluated alongside non-targeted nanobubbles using contrast enhanced ultrasound both in vitro and in vivo in mice. In vitro resonant mass and clinical ultrasound measurements showed gas-core, lipid-shelled nanobubbles conjugated to either a PTPmu-directed peptide or a Scrambled control peptide were equivalent. Mice with heterotopic human tumors expressing the PTPmu-biomarker were injected with PTPmu-targeted or control nanobubbles and dynamic contrast-enhanced ultrasound was performed. Tumor enhancement was more rapid and greater with PTPmu-targeted nanobubbles compared to the non-targeted control nanobubbles. Peak tumor enhancement by the PTPmu-targeted nanobubbles occurred within five minutes of contrast injection and was more than 35% higher than the Scrambled nanobubble signal for the subsequent two minutes. At later time points, the signal in tumors remained higher with PTPmu-targeted nanobubbles demonstrating that PTPmu-targeted nanobubbles recognize tumors using molecular ultrasound imaging and may be useful for diagnostic and therapeutic purposes.

Ultrasound molecular imaging of ovarian cancer with CA-125 targeted nanobubble contrast agents

Ultrasound is frequently utilized in diagnosis of gynecologic malignancies such as ovarian cancer. Because epithelial ovarian cancer (EOC) is often characterized by overexpression of cancer antigen 125 (CA-125), ultrasound contrast agents able to target this molecular signature could be a promising complementary strategy. In this work, we demonstrate application of CA-125-targeted echogenic lipid and surfactant-stabilized nanobubbles imaged with standard clinical contrast harmonic ultrasound for imaging of CA-125 positive OVCAR-3 tumors in mice. Surface functionalization of the nanobubbles with a CA-125 antibody achieved rapid significantly (P < 0.05) enhanced tumor accumulation, higher peak ultrasound signal intensity and slower wash out rates in OVCAR-3 tumors compared to CA-125 negative SKOV-3 tumors. Targeted nanobubbles also exhibited increased tumor retention and prolonged echogenicity compared to untargeted nanobubbles. Data suggest that ultrasound molecular imaging using CA-125 antibody-conjugated nanobubbles may contribute to improved diagnosis of EOC.

Sequential HIFU heating and nanobubble encapsulation provide efficient drug penetration from stealth and temperature sensitive liposomes in colon cancer

Mild hyperthermia generated using high intensity focused ultrasound (HIFU) and microbubbles (MBs) can improve tumor drug delivery from non-thermosensitive liposomes (NTSLs) and low temperature sensitive liposomes (LTSLs). However, MB and HIFU are limited by the half-life of the contrast agent and challenges in accurate control of large volume tumor hyperthermia for longer duration (>30min.). The objectives of this study were to: 1) synthesize and characterized long-circulating echogenic nanobubble encapsulated LTSLs (ELTSLs) and NTSLs (ENTSLs), 2) evaluate in vivo drug release following short duration (~20min each) HIFU treatments administered sequentially over an hour in a large volume of mouse xenograft colon tumor, and 3) determine the impact of the HIFU/nanobubble combination on intratumoral drug distribution. LTSLs and NTSLs containing doxorubicin (Dox) were co-loaded with a nanobubble contrast agent (perfluoropentane, PFP) using a one-step sonoporation method to create ELTSLs and ENTSLs, which then were characterized for size, release in a physiological buffer, and ability to encapsulate PFP. For the HIFU group, mild hyperthermia (40–42°C) was completed within 90min after liposome infusion administered sequentially in three regions of the tumor. Fluorescence microscopy and high performance liquid chromatography analysis were performed to determine the spatial distribution and concentration of Dox in the treated regions. PFP encapsulation within ELTSLs and ENTSLs did not impact size or caused premature drug release in physiological buffer. As time progressed, the delivery of Dox decreased in HIFU-treated tumors with ELTSLs, but this phenomenon was absent in the LTSL, NTSL, and ENTSL groups. Most importantly, PFP encapsulation improved Dox penetration in the tumor periphery and core and did not impact the distribution of Dox in non-tumor organs/tissues. Data from this study suggest that short duration and sequential HIFU treatment could have significant benefits and that its action can be potentiated by nanobubble agents to result in improved drug penetration.

Production and characterization of a novel long-acting Herceptin-targeted nanobubble contrast agent specific for Her-2-positive breast cancers.

BackgroundThere is an unmet need for specific and sensitive imaging techniques to assess the efficacy of breast cancer therapy, particularly Her-2-expressing cancers. Ultrasonic microbubbles are being developed for use as diagnostic and therapeutic tools. However, nanobubbles circulate longer, are smaller, and diffuse into extravascular tissue to specifically bind target molecules. Here, we characterize a novel Herceptin-conjugated nanobubble for use against Her-2-expressing tumors.MethodsPhospholipid-shelled nanobubbles conjugated with Herceptin (NBs-Her) were fabricated using a thin-film hydration method and characterized in vitro in breast cancer cell lines and in vivo in a mouse model.ResultsThe average size of the unconjugated nanobubbles (NBs-Blank) and NBs-Her was 447.1 ± 18.4 and 613.0 ± 25.4 nm, respectively. In cell culture, the NBs-Her adhered to Her-2-positive cells significantly better than to Her-2-negative cells (p < 0.05). In vivo, the peak intensity and the half-time to washout of the NBs-Her were significantly greater than those of the NBs-Blank (p < 0.05). In addition, contrast-enhanced ultrasound imaging quality was improved through the use of the NBs-Her. The nanobubbles were able to penetrate into tumor tissue to allow extravascular imaging, but did not penetrate normal skeletal muscle.ConclusionsThe Herceptin-conjugated nanobubble had many properties that made it useful for in vivo imaging, including longer circulation time and better tumor selectivity. This platform may be able to provide targeted delivery of therapeutic drugs or genes.

X-ray spatial frequency heterodyne imaging of protein-based nanobubble contrast agents.

Spatial Frequency Heterodyne Imaging (SFHI) is a novel x-ray scatter imaging technique that utilizes nanoparticle contrast agents. The enhanced sensitivity of this new technique relative to traditional absorption-based x-ray radiography makes it promising for applications in biomedical and materials imaging. Although previous studies on SFHI have utilized only metal nanoparticle contrast agents, we show that nanomaterials with a much lower electron density are also suitable. We prepared protein-based "nanobubble" contrast agents that are comprised of protein cage architectures filled with gas. Results show that these nanobubbles provide contrast in SFHI comparable to that of gold nanoparticles of similar size.

Acoustic Characterization and Pharmacokinetic Analyses of New Nanobubble Ultrasound Contrast Agents

In contrast to the clinically used microbubble ultrasound contrast agents, nanoscale bubbles (or nanobubbles) may potentially extravasate into tumors that exhibit more permeable vasculature, facilitating targeted molecular imaging and drug delivery. Our group recently presented a simple strategy using the non-ionic surfactant Pluronic as a size control excipient to produce nanobubbles with a mean diameter of 200nm that exhibited stability and echogenicity on par with microbubbles. The objective of this study was to carry out an in-depth characterization of nanobubble properties as compared with Definity microbubbles, both invitro and invivo. Through use of a tissue-mimicking phantom, invitro experiments measured the echogenicity of the contrast agent solutions and the contrast agent dissolution rate over time. Nanobubbles were found to be more echogenic than Definity microbubbles at three different harmonic frequencies (8, 6.2 and 3.5MHz). Definity microbubbles also dissolved 1.67 times faster than nanobubbles. Pharmacokinetic studies were then performed invivo in a subcutaneous human colorectal adenocarcinoma (LS174T) in mice. The peak enhancement and decay rates of contrast agents after bolus injection in the liver, kidney and tumor were analyzed. No significant differences were observed in peak enhancement between the nanobubble and Definity groups in the three tested regions (tumor, liver and kidney). However, the decay rates of nanobubbles in tumor and kidney were significantly slower than those of Definity in the first 200-s fast initial phase. There were no significant differences in the decay rates in the liver in the initial phase or in three regions of interest in the terminal phase. Our results suggest that the stability and acoustic properties of the new nanobubble contrast agents are superior to those of the clinically used Definity microbubbles. The slower washout of nanobubbles in tumors suggests potential entrapment of the bubbles within the tumor parenchyma.