Nonlinear Frequency Mixing Ultrasound Imaging of Nanoscale Contrast Agents.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Nonlinear frequency mixing significantly improved the nanobubble contrast, which facilitated their imaging in-vivo. SIGNIFICANCE: This study offers a new avenue to enhance ultrasound imaging utilizing nanobubbles, potentially leading to advancements in other diagnostic applications.

Nanobubble-mediated cancer cell sonoporation using low-frequency ultrasound.

Ultrasound insonation of microbubbles can form transient pores in cell membranes that enable the delivery of non-permeable extracellular molecules to the cells. Reducing the size of microbubble contrast agents to the nanometer range could facilitate cancer sonoporation. This size reduction can enhance the extravasation of nanobubbles into tumors after an intravenous injection, thus providing a noninvasive sonoporation platform. However, drug delivery efficacy depends on the oscillations of the bubbles, the ultrasound parameters and the size of the target compared to the membrane pores. The formation of large pores is advantageous for the delivery of large molecules, however the small size of the nanobubbles limit the bioeffects when operating near the nanobubble resonance frequency at the MHz range. Here, we show that by coupling nanobubbles with 250 kHz low frequency ultrasound, high amplitude oscillations can be achieved, which facilitate low energy sonoporation of cancer cells. This is beneficial both for increasing the uptake of a specific molecule and to improve large molecule delivery. The method was optimized for the delivery of four fluorescent molecules ranging in size from 1.2 to 70 kDa to breast cancer cells, while comparing the results to targeted microbubbles. Depending on the fluorescent molecule size, the optimal ultrasound peak negative pressure was found to range between 300 and 500 kPa. Increasing the pressure to 800 kPa reduced the fraction of fluorescent cells for all molecules sizes. The optimal uptake for the smaller molecule size of 4 kDa resulted in a fraction of 19.9 ± 1.8% of fluorescent cells, whereas delivery of 20 kDa and 70 kDa molecules yielded 14 ± 0.8% and 4.1 ± 1.1%, respectively. These values were similar to targeted microbubble-mediated sonoporation, suggesting that nanobubbles can serve as noninvasive sonoporation agents with a similar potency, and at a reduced bubble size. The nanobubbles effectively reduced cell viability and may thus potentially reduce the tumor burden, which is crucial for the success of cancer treatment. This method provides a non-invasive and low-energy tumor sonoporation theranostic platform, which can be combined with other therapies to maximize the therapeutic benefits of cancer treatment or be harnessed in gene therapy applications.

Low frequency nanobubble-enhanced ultrasound mechanotherapy for noninvasive cancer surgery.

Scaling down the size of microbubble contrast agents to the nanometer level holds the promise for noninvasive cancer therapy. However, the small size of nanobubbles limits the obtained bioeffects as a result of ultrasound cavitation, when operating near the nanobubble resonance frequency. Here we show that coupled with low energy insonation at a frequency of 80 kHz, well below the resonance frequency of these agents, nanobubbles serve as noninvasive therapeutic warheads that trigger potent mechanical effects in tumors following a systemic injection. We demonstrate these capabilities in tissue mimicking phantoms, where a comparison of the acoustic response of micro- and nano-bubbles after insonation at a frequency of 250 or 80 kHz revealed that higher pressures were needed to implode the nanobubbles compared to microbubbles. Complete nanobubble destruction was achieved at a mechanical index of 2.6 for the 250 kHz insonation vs. 1.2 for the 80 kHz frequency. Thus, the 80 kHz insonation complies with safety regulations that recommend operation below a mechanical index of 1.9. In vitro in breast cancer tumor cells, the cell viability was reduced to 17.3 ± 1.7% of live cells. In vivo, in a breast cancer tumor mouse model, nanobubble tumor distribution and accumulation were evaluated by high frequency ultrasound imaging. Finally, nanobubble-mediated low frequency insonation of breast cancer tumors resulted in effective mechanical tumor ablation and tumor tissue fractionation. This approach provides a unique theranostic platform for safe, noninvasive and low energy tumor mechanotherapy.

Acoustically Detonated Microbubbles Coupled with Low Frequency Insonation: Multiparameter Evaluation of Low Energy Mechanical Ablation.

Noninvasive ultrasound surgery can be achieved using focused ultrasound to locally affect the targeted site without damaging intervening tissues. Mechanical ablation and histotripsy use short and intense acoustic pulses to destroy the tissue via a purely mechanical effect. Here, we show that coupled with low-frequency excitation, targeted microbubbles can serve as mechanical therapeutic warheads that trigger potent mechanical effects in tumors using focused ultrasound. Upon low frequency excitation (250 kHz and below), high amplitude microbubble oscillations occur at substantially lower pressures as compared to higher MHz ultrasonic frequencies. For example, inertial cavitation was initiated at a pressure of 75 kPa for a center frequency of 80 kHz. Low frequency insonation of targeted microbubbles was then used to achieve low energy tumor cell fractionation at pressures below a mechanical index of 1.9, and in accordance with the Food and Drug Administration guidelines. We demonstrate these capabilities in vitro and in vivo. In cell cultures, cell viability was reduced to 16% at a peak negative pressure of 800 kPa at the 250 kHz frequency (mechanical index of 1.6) and to 10% at a peak negative pressure of 250 kPa at a frequency of 80 kHz (mechanical index of 0.9). Following an intratumoral injection of targeted microbubbles into tumor-bearing mice, and coupled with low frequency ultrasound application, significant tumor debulking and cancer cell death was observed. Our findings suggest that reducing the center frequency enhances microbubble-mediated mechanical ablation; thus, this technology provides a unique theranostic platform for safe low energy tumor fractionation, while reducing off-target effects.

Real-time detection of copper contaminants in environmental water using porous silicon Fabry-Pérot interferometers.

Water sources are vulnerable to intentional and inadvertent human pollution with thousands of synthetic and geogenic trace contaminants, posing long-term effects on the aquatic ecosystem and human health. Thus, early and rapid detection of water pollutants followed by corrective and preventive actions can lead to the reduction of the overall polluting impact to safeguard public health. This study presents a generic sensing assay for the label-free detection of copper contaminants in environmental water samples using multilayered polyethylenimine (PEI) functionalized porous silicon Fabry-Pérot interferometers. The selective chelating activity of PEI thin-films was monitored in real-time by reflective interferometric Fourier transform spectroscopy (RIFTS) while assessing the improved optical responses. The optimized scaffold of two sequential PEI layers depicted a linear working range between 0.2 and 2 ppm while presenting a detection limit of 0.053 ppm (53 ppb). The specificity of the developed platform was cross-validated against various metallic pollutants and cations commonly found in water bodies (i.e., Cd2+, Pb2+, Cr3+, Fe3+, Mg2+, Ca2+, Zn2+, K+ and Al3+). Finally, as a proof of concept, the analytical performance of the porous interferometers for real-life scenarios was demonstrated in three water samples (tap, ground and irrigation), presenting sufficient adaptability to complex matrix analysis with recovery values of 85-106%. Overall, the developed sensing concept offers an efficient, rapid and label-free methodology that can be potentially adopted for routine on-site detection using a simple and portable device.