Chung-Hsin Wang

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.

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.

Superparamagnetic iron oxide and drug complex-embedded acoustic droplets for ultrasound targeted theranosis.

Ultrasound-triggered acoustic droplet vaporization (ADV) has been reported as a mechanical and chemical theranostic strategy for tumor treatment. However, targeting of sufficient amounts of droplets to solid tumors to direct effective mechanical force toward tumor cells remains a major challenge. In this study, we incorporated superparamagnetic iron oxide (SPIO) nanoparticles into acoustic droplets to allow both magnetism-assisted targeting and magnetic resonance (MR)-guided ultrasound-triggered ADV. The multi-functionality of these droplets was further increased by co-encapsulation of the chemotherapeutic drug doxorubicin (DOX) and surface conjugation of anti-vascular endothelial growth factor receptor 2 antibody, to serve as an additional targeting moiety. Maximum loading capacities of 7.69 mg SPIO and 1.53 mg DOX per mL were achieved, and magnetic properties were characterized by determination of magnetic hysteresis curves and transverse relaxation rates. In vitro and in vivo MR imaging demonstrated the feasibility of dual modal imaging of SPIO-embedded droplets. Finally, a vessel-mimicking phantom model with live C6 glioma cells was used to demonstrate a 5.4-fold improvement in targeting efficacy by magnetism-assisted targeting of the SPIO-embedded droplets, and effective disruption of cells by insonation-induced ADV, suggesting the potential of developing this system for future clinical applications.

Biomimetic Acoustically-Responsive Vesicles for Theranostic Applications

In recent years, biomimetic cell membrane-derived particles have emerged as a new class of drug delivery system with advantages of biocompatibility, ease of isolation and long circulation profile. Here we report the development and potential theranostic applications of a new biomimetic acoustically-responsive droplet system derived from mammalian red blood cell membrane (RBCM). We hypothesized that drug-loaded RBCM droplets (RBCMDs) would undergo a transition from liquid (droplets) to gas (bubbles) upon high intensity focused ultrasound (HIFU) insonation, resulting in on-demand drug release. The generated microbubbles could also serve as a contrast agent to enhance ultrasound imaging. As-synthesized RBCMDs exhibited uniform size, good dispersity and preservation of RBCM-associated proteins that prevented uptake by macrophages. Camptothecin (CPT), an anti-cancer drug, was successfully loaded in the RBCMDs with a loading efficiency of 2-3% and an encapsulation efficiency of 62-97%. A short (3 min) exposure to HIFU irradiation triggered release of CPT from the RBCMDs and the physical explosion of droplets damaged nearby cancer cells resulting in significant cell death. In addition, the acoustically vaporized RBCMDs significantly increased the ultrasound echo signal to 30 dB. Lastly, we demonstrated that RBCMDs could be acoustically vaporized in vivo in target tissues, and enhancing ultrasound imaging. Taken together, we have developed a new class of naturally derived RBCMDs which show great potential for future application in remotely triggered drug delivery and ultrasound imaging enhancement.

Internal polymer scaffolding in lipid-coated microbubbles for control of inertial cavitation in ultrasound theranostics

A lipid-polymer composite structure was developed for tuning of inertial cavitation activity of microbubbles under ultrasound exposure. The incorporation of a thin layer of polymer networks inside the lipid monolayer resulted in marked reduction in the inertial cavitation dose. This strategy has the potential to increase the safety of ultrasound theranostic applications assisted by microbubble cavitation.

Feasibility study of using macrophages as drug delivery carriers for drug-loaded phase-change droplets

Tumors normally possess irregular vasculature, and tend to outstrip blood supplement and become hypoxia or ischemia, resulting in the resistances of the tumor to conventional chemotherapy and radiotherapy. New therapies are required to target hypoxic or ischemic areas in tumors. In this study, we investigate the feasibility of using macrophages to infiltrate hypoxic or ischemic areas in tumors, as the carriers of drug-loaded phase-change droplets. RAW 264.7 cells (mouse leukaemic monocyte macrophage cell line) were used to ingest the drug-loaded phase-change droplets. The droplets were composed of lipid, perfluoropentane (PFP) and chemotherapeutic drug - doxorubicin (DOX). Fluorescence spectrophotometer was used to quantify the drug encapsulation efficiency. For evaluating DOX-droplets uptake efficiency, cell viability and migration mobility of DOX-droplet loaded macrophages, flow cytometric analysis, Alamar Blue assay, and transmembrane cell migration assay were measured, respectively. The results demonstrate the feasibility of using macrophages to deliver DOX-loaded phase-change droplets. Ultrasound-triggered vaporization was also performed to investigate the drug liberation efficiency from the droplet-loaded macrophages. Future works include the assessments of the tumor infiltration ability of droplet-loaded macrophages and the liberated drug payload via ultrasound-triggered vaporization in vivo.

Real-time monitoring of inertial cavitation effects of microbubbles by using MRI: In vitro experiments

To investigate the feasibility of half‐Fourier acquisition single‐shot turbo spin‐echo (HASTE) for real‐time monitoring of signal changes because of water flow induced by inertial cavitation (IC) during microbubbles (MBs)‐present focused ultrasound (FUS) exposure.

One-step covalently conjugated aptamer microbubbles for ultrasound targeted imaging

In this study, we purpose a covalently conjugated microbubbles coupling with nucleic acid ligands, aptamers, for providing a higher specific affinity for ultrasound targeting studies. The sgc8c aptamer was linked with bubbles through thiol-maleimide coupling chemistry for specific targeting to CCRF-CEM cells. Several investigations were used to discuss the performance and consistency of the prepared bubbles, such as size distribution, conjugation efficiency analysis and flow cytometry assay. Further, we applied our conjugated bubbles to ex-vivo ultrasound targeted imaging and compared with optical images.

Macrophages as Drug Delivery Carriers for Acoustic Phase-Change Droplets

The major challenges in treating malignant tumors are transport of therapeutic agents to hypoxic regions and real-time assessment of successful drug release via medical imaging modalities. In this study, we propose the use of macrophages (RAW 264.7 cells) as carriers of drug-loaded phase-change droplets to penetrate ischemic or hypoxic regions within tumors. The droplets consist of perfluoropentane, lipid and the chemotherapeutic drug doxorubicin (DOX, DOX-droplets). The efficiency of DOX-droplet uptake, migration mobility and viability of DOX-droplet-loaded macrophages (DLMs) were measured using a transmembrane cell migration assay, the alamarBlue assay and flow cytometric analysis, respectively. Our results indicate the feasibility of utilizing macrophages as DOX-droplet carriers (DOX payload of DOX-droplets: 459.3 ± 35.8 µg/mL, efficiency of cell uptake DOX-droplets: 88.8 ± 3.5%). The migration mobility (total number of migrated microphages) of DLMs decreased to 32.3% compared with that of healthy macrophages, but the DLMs provided contrast-enhanced ultrasound imaging (1.7-fold enhancement) and anti-tumor effect (70.9% cell viability) after acoustic droplet vaporization, suggesting the potential theranostic applications of DLMs. Future work will assess the tumor penetration ability of DLMs, mechanical effect of droplet vaporization on in vivo anti-tumor therapy and the release of the carried drug by ultrasound-triggered vaporization.