Shih-Tsung Kang

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.

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.

Ultrasound microbubble contrast agents for diagnostic and therapeutic applications: current status and future design.

Ultrasound contrast agents are highly echogenic microbubbles with many unique properties. Microbubbles can basically improve the sensitivity of conventional ultrasound imaging to the microcirculation. The resonance of microbubbles in response to an incident ultrasound pulse results in nonlinear harmonic emission that serves as the signature of microbubbles in microbubble-specific imaging. Inertial cavitation and destruction of microbubbles can produce a strong mechanical stress enhancing the permeability of the surrounding tissues, and can further increase the extravasation of drugs from the blood into the cytoplasm or interstitium. Stable cavitation by high-frequency ultrasound can also mildly increase tissue permeability without causing any damage even at a high acoustic pressure. Microbubbles can carry drugs, release them upon ultrasound-mediated microbubble destruction, and simultaneously enhance vascular permeability to increase drug deposition in tissues. Various targeting ligands can be conjugated to the surface of microbubbles to attain ligand-directed and site-specific accumulation for targeted imaging. In addition to current developments in microbubble technology, this review introduces our studies of the applications of microbubble- specific imaging, ultrasound-aided drug delivery, and targeted imaging. These applications are promising but may require further improvement for clinical use.

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.

Mechanical bioeffects of acoustic droplet vaporization in vessel-mimicking phantoms

This study investigated the mechanical bioeffects exerted by acoustic droplet vaporization (ADV) under different experimental conditions using vessel phantoms with a 200-μm inner diameter but different stiffness for imitating the microvasculature in various tumors. High-speed microscopy, passive cavitation detection, and ultrasound attenuation measurement were conducted to determine the morphological characteristics of vascular damage and clarify the mechanisms by which the damage was initiated and developed. The results show that phantom erosion was initiated under successive ultrasound exposure (2 MHz, 3 cycles) at above 8-MPa peak negative pressures (PNPs) when ADV occurred with inertial cavitation (IC), producing lesions whose morphological characteristics were dependent on the amount of vaporized droplets. Slight injury occurred at droplet concentrations below (2.6±0.2)×10(6) droplets/mL, forming shallow and rugged surfaces on both sides of the vessel walls. Increasing the droplet concentration to up to (2.6±0.2)×10(7) droplets/mL gradually suppressed the damage on the distal wall, and turned the rugged surface on the proximal wall into tunnels rapidly elongating in the direction opposite to ultrasound propagation. Increasing the PNP did not increase the maximum tunnel depth after the ADV efficiency reached a plateau (about 71.6±2.7% at 10 MPa). Increasing the pulse duration effectively increased the maximum tunnel depth to more than 10 times the diameter of the vessel even though there was no marked enhancement in IC dose. It can be inferred that substantial bubble generation in single ADV events may simultaneously distort the acoustic pressure distribution. The backward ultrasound reinforcement and forward ultrasound shielding relative to the direction of wave propagation augment the propensity of backward erosion. The results of the present work provide information that is valuable for the prevention or utilization of ADV-mediated mechanical bioeffects in clinical applications.

A maleimide-based in-vitro model for ultrasound targeted imaging

Intricate variations and poor visual access result in the difficulty in studying the property of adherent targeted bubbles using an in vivo model. Here, we propose a simple in-vitro model based on the natural adhesion of maleimide bubbles to gelatin. We validated the maleimide-mediated bubble adhesion using flat gelatin phantoms. Treating the gelatin surfaces with reducing agent yielded abundant cysteine molecules for attaching maleimide bubbles. An optical microscope and a homemade ultrasound imaging system equipped with a 40-MHz transducer were adopted to observe the acoustic responses of adherent bubbles. Comparing the results of optical observations from experimental and control tests support the bubble adhesion indeed relying on maleimide-cysteine tethering. The intensity of the echoes from a bubble-bound gelatin surface increased with the bubble adhesion density compared with that from a clear gelatin surface. The echo enhancement reached a plateau at 40-42 dB as the bubble adhesion densities were higher than 1.47 × 10(5) bubbles/mm(2). The adherent bubbles would be disrupted rapidly under the exposure of 300-kPa ultrasound pulses. However, increasing the adhesion density to 3.62 × 10(5) bubbles/mm(2) resulted in the echo enhancement being maintained at a duration of 40 min. The advantages of this in-vitro model over previously proposed ones include better stability, less expense, and fewer preparation procedures.

Focused Ultrasound-Induced Blood-Brain Barrier Opening: Association with Mechanical Index and Cavitation Index Analyzed by Dynamic Contrast-Enhanced Magnetic-Resonance Imaging.

Focused ultrasound (FUS) with microbubbles can temporally open the blood-brain barrier (BBB), and the cavitation activities of microbubbles play a key role in the BBB-opening process. Previous attempts used contrast-enhanced magnetic resonance imaging (CE-MRI) to correlate the mechanical index (MI) with the scale of BBB-opening, but MI only partially gauged acoustic activities, and CE-MRI did not fully explore correlations of pharmacodynamic/pharmacokinetic behaviors. Recently, the cavitation index (CI) has been derived to serve as an indicator of microbubble-ultrasound stable cavitation, and may also serve as a valid indicator to gauge the level of FUS-induced BBB opening. This study investigates the feasibility of gauging FUS-induced BBB opened level via the two indexes, MI and CI, through dynamic contrast-enhanced (DCE)-MRI analysis as well as passive cavitation detection (PCD) analysis. Pharmacodynamic/pharmacokinetic parameters derived from DCE-MRI were characterized to identify the scale of FUS-induced BBB opening. Our results demonstrated that DCE-MRI can successfully access pharmacodynamic/pharmacokinetic BBB-opened behavior, and was highly correlated both with MI and CI, implying the feasibility in using these two indices to gauge the scale of FUS-induced BBB opening. The proposed finding may facilitate the design toward using focused ultrasound as a safe and reliable noninvasive CNS drug delivery.

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.

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.

Inertial cavitation initiated by polytetrafluoroethylene nanoparticles under pulsed ultrasound stimulation

Nanoscale gas bubbles residing on a macroscale hydrophobic surface have a surprising long lifetime (on the order of days) and can serve as cavitation nuclei for initiating inertial cavitation (IC). Whether interfacial nanobubbles (NBs) reside on the infinite surface of a hydrophobic nanoparticle (NP) and could serve as cavitation nuclei is unknown, but this would be very meaningful for the development of sonosensitive NPs. To address this problem, we investigated the IC activity of polytetrafluoroethylene (PTFE) NPs, which are regarded as benchmark superhydrophobic NPs due to their low surface energy caused by the presence of fluorocarbon. Both a passive cavitation detection system and terephthalic dosimetry was applied to quantify the intensity of IC. The IC intensities of the suspension with PTFE NPs were 10.30 and 48.41 times stronger than those of deionized water for peak negative pressures of 2 and 5MPa, respectively. However, the IC activities were nearly completely inhibited when the suspension was degassed or ethanol was used to suspend PTFE NPs, and they were recovered when suspended in saturated water, which may indicates the presence of interfacial NBs on PTFE NPs surfaces. Importantly, these PTFE NPs could sustainably initiate IC for excitation by a sequence of at least 6000 pulses, whereas lipid microbubbles were completely depleted after the application of no more than 50 pulses under the same conditions. The terephthalic dosimetry has shown that much higher hydroxyl yields were achieved when PTFE NPs were present as cavitation nuclei when using ultrasound parameters that otherwise did not produce significant amounts of free radicals. These results show that superhydrophobic NPs may be an outstanding candidate for use in IC-related applications.