Chih-Kuang Yeh

Concurrent blood-brain barrier opening and local drug delivery using drug-carrying microbubbles and focused ultrasound for brain glioma treatment.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor. The blood-brain barrier (BBB) provides a major obstacle to chemotherapy since therapeutic doses cannot be achieved by traditional drug delivery without severe systemic cytotoxic effects. Recently, microbubble (MB)-enhanced focused ultrasound (FUS) was shown to temporally and locally disrupt the BBB thereby enhancing drug delivery into brain tumors. Here we propose the concept of smart, multifunctional MBs capable of facilitating FUS-induced BBB disruption while serving as drug-carrying vehicles and protecting drugs from rapid degradation. The designed MBs had a high loading capacity (efficiency of 68.01 ± 4.35%) for 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU). When combined with FUS (1-MHz), these BCNU-MBs facilitated local BBB disruption and simultaneously released BCNU at the target site, thus increasing local BCNU deposition. Encapsulation of BCNU in MBs prolonged its circulatory half-life by 5-fold, and accumulation of BCNU in the liver was reduced 5-fold due to the slow reticuloendothelial system uptake of BCNU-MBs. In tumor-bearing animals, BCNU-MBs with FUS controlled tumor progression (915.3%-39.6%) and improved median survival (29 days-32.5 days). This study provides a new approach for designing multifunctional MBs to facilitate FUS-mediated chemotherapy for brain tumor treatment.

SPIO-conjugated, doxorubicin-loaded microbubbles for concurrent MRI and focused-ultrasound enhanced brain-tumor drug delivery

The blood-brain barrier (BBB) can be temporarily and locally opened by focused ultrasound (FUS) in the presence of circulating microbubbles (MBs). Currently, contrast-enhanced magnetic resonance imaging (CE-MRI) is used to monitor contrast agent leakage to verify BBB-opening and infer drug deposition. However, despite being administered concurrently, MBs, therapeutic agent, and contrast agent have distinct pharmacodynamic behaviors, thus complicating the quantification and optimization of BBB-opening and drug delivery. Here we propose multifunctional MBs loaded with therapeutic agent (doxorubicin; DOX) and conjugated with superparamagnetic iron oxide (SPIO) nanoparticles. These DOX-SPIO-MBs were designed to concurrently open the BBB and perform drug delivery upon FUS exposure, act as dual MRI and ultrasound contrast agent, and allow magnetic targeting (MT) to achieve enhanced drug delivery. We performed burst-tone FUS after injection of DOX-SPIO-MBs, followed by MT with an external magnet attached to the scalp in a rat glioma model. Animals were monitored by T2-weighted MRI and susceptibility weighted imaging and the concentration of SPIO particles was determined by spin-spin relaxivity. We found that DOX-SPIO-MBs were stable and provided significant superparamagnetic/acoustic properties for imaging. BBB-opening and drug delivery were achieved concurrently during the FUS exposure. In addition, MT increased local SPIO deposition in tumor regions by 22.4%. Our findings suggest that DOX-SPIO-MBs with FUS could be an excellent theranostic tool for future image-guided drug delivery to brain tumors.

Aptamer-conjugated and drug-loaded acoustic droplets for ultrasound theranosis

Tumor therapy requires multi-functional treatment strategies with specific targeting of therapeutics to reduce general toxicity and increase efficacy. In this study we fabricated and functionally tested aptamer-conjugated and doxorubicin (DOX)-loaded acoustic droplets comprising cores of liquid perfluoropentane compound and lipid-based shell materials. Conjugation of sgc8c aptamers provided the ability to specifically target CCRF-CEM cells for both imaging and therapy. High-intensity focused ultrasound (HIFU) was introduced to trigger targeted acoustic droplet vaporization (ADV) which resulted in both mechanical cancer cell destruction by inertial cavitation and chemical treatment through localized drug release. HIFU insonation showed a 56.8% decrease in cell viability with aptamer-conjugated droplets, representing a 4.5-fold increase in comparison to non-conjugated droplets. In addition, the fully-vaporized droplets resulted in the highest DOX uptake by cancer cells, compared to non-vaporized or partially vaporized droplets. Optical studies clearly illustrated the transient changes that occurred upon ADV of droplet-targeted CEM cells, and B-mode ultrasound imaging revealed contrast enhancement by ADV in ultrasound images. In conclusion, our fabricated droplets functioned as a hybrid chemical and mechanical strategy for the specific destruction of cancer cells upon ultrasound-mediated ADV, while simultaneously providing ultrasound imaging capability.

Combining microbubbles and ultrasound for drug delivery to brain tumors: current progress and overview.

Malignant glioma is one of the most challenging central nervous system (CNS) diseases, which is typically associated with high rates of recurrence and mortality. Current surgical debulking combined with radiation or chemotherapy has failed to control tumor progression or improve glioma patient survival. Microbubbles (MBs) originally serve as contrast agents in diagnostic ultrasound but have recently attracted considerable attention for therapeutic application in enhancing blood-tissue permeability for drug delivery. MB-facilitated focused ultrasound (FUS) has already been confirmed to enhance CNS-blood permeability by temporally opening the blood-brain barrier (BBB), thus has potential to enhance delivery of various kinds of therapeutic agents into brain tumors. Here we review the current preclinical studies which demonstrate the reports by using FUS with MB-facilitated drug delivery technology in brain tumor treatment. In addition, we review newly developed multifunctional theranostic MBs for FUS-induced BBB opening for brain tumor therapy.

Paclitaxel-liposome–microbubble complexes as ultrasound-triggered therapeutic drug delivery carriers

Liposome-microbubble complexes (LMC) have become a promising therapeutic carrier for ultrasound-triggered drug delivery to treat malignant tumors. However, the efficacy for ultrasound-assisted chemotherapy in vivo and the underlying mechanisms remain to be elucidated. Here, we investigated the feasibility of using paclitaxel-liposome-microbubble complexes (PLMC) as possible ultrasound (US)-triggered targeted chemotherapy against breast cancer. PTX-liposomes (PL) were conjugated to the microbubble (MB) surface through biotin-avidin linkage, increasing the drug-loading efficiency of MBs. The significant increased release of payloads from liposome-microbubble complexes was achieved upon US exposure. We used fluorescent quantum dots (QDs) as a model drug to show that released QDs were taken up by 4T1 breast cancer cells treated with QD-liposome-microbubble complexes (QLMC) and US, and uptake depended on the exposure time and intensity of insonication. We found that PLMC plus US inhibited tumor growth more effectively than PL plus US or PLMC without US, not only in vitro, but also in vivo. Histologically, the inhibition of tumor growth appeared to result from increased apoptosis and reduced angiogenesis in tumor xenografts. In addition, a significant increase of drug concentration in tumors was observed in comparison to treatment with non-conjugated PL or PLMC without US. The significant increase in an antitumor efficacy of PLMC plus US suggests their potential use as a new targeted US chemotherapeutic approach to inhibit breast cancer growth.

A new imaging strategy utilizing wideband transient response of ultrasound contrast agents

High-resolution clinical systems operating near 15 MHz are becoming more available; however, they lack sensitive harmonic imaging modes for ultrasound contrast agent (UCA) detection, primarily due to limited bandwidth. When a UCA is driven to nonlinear oscillation, a very wideband acoustic transient response is produced that extends beyond 15 MHz. We propose a novel strategy using two separate transducers at widely separated frequencies and arranged confocally to simultaneously excite and receive acoustic transients from UCAs. Experiments were performed to demonstrate that this new mode shows similar resolution, higher echo amplitudes, and greatly reduced attenuation compared to transmission at a higher frequency, and superior resolution compared to transmission and reception at a lower frequency. The proposed method is shown to resolve two 200 /spl mu/m tubes with centers separated by 400//spl mu/m. Strong acoustic transients were detected for rarefaction-first 1-cycle pulses with peak-negative pressures above 300 kPa. The results of this work may lead to uses in flow and/or targeted imaging in applications requiring very high sensitivity to contrast agents.

Antiangiogenic-targeting drug-loaded microbubbles combined with focused ultrasound for glioma treatment

Current chemotherapeutic agents do not only kill tumor cells but also induce systemic toxicity that significantly limits their dosage. Focused ultrasound (FUS) in the presence of microbubbles (MBs) is capable of transient and local opening of the blood-brain barrier (BBB) that enhances chemotherapeutic drug delivery into the brain parenchyma for glioma treatment. Our previous results demonstrated the success of combining the use of drug (1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU)-loaded MBs with FUS-induced BBB opening to improve local drug delivery and reduce systemic toxicity. Here we introduce novel VEGF-targeting, drug-loaded MBs that significantly further enhance targeted drug release and reduce tumor progression in a rat model, using the FUS-BBB opening strategy. This study suggests a promising direction for future MB design aimed at targeted brain tumor therapy, and the possible future extension of MB application towards theragnostic use.

Aptamer-Conjugated Nanobubbles for Targeted Ultrasound Molecular Imaging

Targeted ultrasound contrast agents can be prepared by some specific bioconjugation techniques. The biotin-avidin complex is an extremely useful noncovalent binding system, but the system might induce immunogenic side effects in human bodies. Previous proposed covalently conjugated systems suffered from low conjugation efficiency and complex procedures. In this study, we propose a covalently conjugated nanobubble coupling with nucleic acid ligands, aptamers, for providing a higher specific affinity for ultrasound targeting studies. The sgc8c aptamer was linked with nanobubbles through thiol-maleimide coupling chemistry for specific targeting to CCRF-CEM cells. Further improvements to reduce the required time and avoid the degradation of nanobubbles during conjugation procedures were also made. Several investigations were used to discuss the performance and consistency of the prepared nanobubbles, such as size distribution, conjugation efficiency analysis, and flow cytometry assay. Further, we applied our conjugated nanobubbles to ex vivo ultrasound targeted imaging and compared the resulting images with optical images. The results indicated the availability of aptamer-conjugated nanobubbles in targeted ultrasound imaging and the practicability of using a highly sensitive ultrasound system in noninvasive biological research.

Classification of Benign and Malignant Breast Tumors by 2-D Analysis Based on Contour Description and Scatterer Characterization

Ultrasound B-mode scanning based on the echo intensity has become an important clinical tool for routine breast screening. The efficacy of the Nakagami parametric image based on the distribution of the backscattered signals for quantifying properties of breast tissue was recently evaluated. The B-mode and Nakagami images reflect different physical characteristic of breast tumors: the former describes the contour features, and the latter reflects the scatterer arrangement inside a tumor. The functional complementation of these two images encouraged us to propose a novel method of 2-D analysis based on describing the contour using the B-mode image and the scatterer properties using the Nakagami image, which may provide useful clues for classifying benign and malignant tumors. To validate this concept, raw data were acquired from 60 clinical cases, and five contour feature parameters (tumor circularity, standard deviation of the normalized radial length, area ratio, roughness index, and standard deviation of the shortest distance) and the Nakagami parameters of benign and malignant tumors were calculated. The receiver operating characteristic curve and fuzzy c-means clustering were used to evaluate the performances of combining the parameters in classifying tumors. The clinical results demonstrated the presence of a tradeoff between the sensitivity and specificity when either using a single parameter or combining two contour parameters to discriminate between benign and malignant cases. However, combining the contour parameters and the Nakagami parameter produces sensitivity and specificity that simultaneously exceed 80%, which means that the functional complementation from the B-scan and the Nakagami image indeed enhances the performance in diagnosing breast tumors.

Intracellular Acoustic Droplet Vaporization in a Single Peritoneal Macrophage for Drug Delivery Applications

This study investigated the acoustic droplet vaporization (ADV) of perfluoropentane (PFP) droplets in single droplet-loaded macrophages (DLMs) by insonation with single three-cycle ultrasound pulses. Transient responses of intracellular ADV within a single DLM were observed with synchronous high-speed photography and cavitation detection. Ultrasound B-mode imaging was further applied to demonstrate the contrast enhancement of ADV-generated bubbles from a group of DLMs. The PFP droplets incorporated in a DLM can be liberated from the cell body after being vaporized into gas bubbles. Inertial cavitation can be simultaneously induced at the same time that bubbles appear. The coalescence of bubbles occurring at the onset of vaporization may facilitate gas embolotherapy and ultrasound imaging. Macrophages can be potential carriers transporting PFP droplets to avascular and hypoxic regions in tumors for ultrasound-controlled drug release and ADV-based tumor therapies.