Qiaofeng Jin

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.

Therapeutic ultrasonic microbubbles carrying paclitaxel and LyP-1 peptide: preparation, characterization and application to ultrasound-assisted chemotherapy in breast cancer cells.

The aim of this work was to develop a novel targeted drug-loaded microbubble (MB) and to investigate its chemotherapy effect in vitro. Paclitaxel (PTX)-loaded lipid MBs were prepared by a mechanical vibration technique. The LyP-1, a breast tumor homing peptide, was coated onto the surface of PTX-loaded MBs through biotin-avidin linkage. The resulting targeted drug-loaded MBs were characterized and applied to ultrasound-assisted chemotherapy in breast cancer cells. Our results showed the ultrasonic MBs were able to achieve 43%-63% of drug encapsulation efficiency, depending on drug loading amount. The binding affinity assay indicated the attachment of targeted MBs to human MDA-MB-231 breast cancer cells was highly efficient and stable even with ultrasonic irradiation on. The cellular uptake efficiency of payload in targeted MBs was 3.71-, 4.95-, 7.43- and 7.66-fold higher than that of non-targeted MBs at the applied ultrasound time of 30, 60, 90 and 120 s, respectively. In addition, the cell proliferation inhibition assay showed the cell viability of targeted PTX-loaded MBs was significantly lower than that of non-targeted PTX-loaded MBs and non-targeted unloaded MBs when ultrasound was utilized. In conclusion, the study indicated the LyP-1-coated PTX-loaded MBs significantly increased the antitumor efficacy and can be used as a potential chemotherapy approach for ultrasound-assisted breast cancer treatment.

Transportation of single cell and microbubbles by phase-shift introduced to standing leaky surface acoustic waves

A microfluidic device was developed to precisely transport a single cell or multiple microbubbles by introducing phase-shifts to a standing leaky surface acoustic wave (SLSAW). The device consists of a polydimethyl-siloxane (PDMS) microchannel and two phase-tunable interdigital transducers (IDTs) for the generation of the relative phase for the pair of surface acoustic waves (SAW) propagating along the opposite directions forming a standing wave. When the SAW contacts the fluid medium inside the microchannel, some of SAW energy is coupled to the fluid and the SAW becomes the leaky surface wave. By modulating the relative phase between two IDTs, the positions of pressure nodes of the SLSAW in the microchannel change linearly resulting in the transportation of a single cell or microbubbles. The results also reveal that there is a good linear relationship between the relative phase and the displacement of a single cell or microbubbles. Furthermore, the single cell and the microbubbles can be transported over a predetermined distance continuously until they reach the targeted locations. This technique has its distinct advantages, such as precise position-manipulation, simple to implement, miniature size, and noninvasive character, which may provide an effective method for the position-manipulation of a single cell and microbubbles in many biological and biomedical applications.

Reversal of multidrug resistance phenotype in human breast cancer cells using doxorubicin-liposome-microbubble complexes assisted by ultrasound

The circumvention of multidrug resistance (MDR) plays a critically important role in the success of chemotherapy. The aim of this work is to investigate the effectiveness and possible mechanisms of the reversal of MDR phenotype in human breast cancer cells by using doxorubicin-liposome-microbubble complexes (DLMC) assisted by ultrasound (US). DLMC is fabricated through conjugating doxorubicin (DOX)-liposome (DL) to the surface of microbubbles (MBs) via the biotin-avidin linkage. The resulting drug-loaded complexes are then characterized and incubated with MCF-7/ADR human breast cancer cells and followed by US exposure. Our results show the more rapid cellular uptake, evident enhancement of nuclear accumulation and less drug efflux in the resistant cells treated by DLMC+US than those treated by DL, DL+verapamil under the same US treatment or DLMC without US. The enhanced drug delivery and cellular uptake also associated with the increase of cytotoxicity against MCF-7/ADR cells, lower MCF-7/ADR cell viability and higher apoptotic cells. Mechanism investigations further disclose a significant increase of reactive oxygen species (ROS) level, enhanced DNA damage and obvious reduction of P-glycoprotein expression in the resistant cells treated with DLMC+US compared with the control cases of cells treated by DLMC, DL+US or DL+verapamil+US. In conclusion, our study demonstrates that DLMC in combination with US may provide an effective delivery of drug to sensitize cells to circumvent MDR and to enhance the therapeutic index of the chemotherapy.

Measurement of flow velocity fields in small vessel-mimic phantoms and vessels of small animals using micro ultrasonic particle image velocimetry (micro-EPIV).

Determining a multidimensional velocity field within microscale opaque fluid flows is needed in areas such as microfluidic devices, biofluid mechanics and hemodynamics research in animal studies. The ultrasonic particle image velocimetry (EchoPIV) technique is appropriate for measuring opaque flows by taking advantage of PIV and B-mode ultrasound contrast imaging. However, the use of clinical ultrasound systems for imaging flows in small structures or animals has limitations associated with spatial resolution. This paper reports on the development of a high-resolution EchoPIV technique (termed as micro-EPIV) and its application in measuring flows in small vessel-mimic phantoms and vessels of small animals. Phantom experiments demonstrate the validity of the technique, providing velocity estimates within 4.1% of the analytically derived values with regard to the flows in a small straight vessel-mimic phantom, and velocity estimates within 5.9% of the computationally simulated values with regard to the flows in a small stenotic vessel-mimic phantom. Animal studies concerning arterial and venous flows of living rats and rabbits show that the micro-EPIV-measured peak velocities within several cardiac cycles are about 25% below the values measured by the ultrasonic spectral Doppler technique. The micro-EPIV technique is able to effectively measure the flow fields within microscale opaque fluid flows.

REAL-TIME TEXTURE ANALYSIS FOR IDENTIFYING OPTIMUM MICROBUBBLE CONCENTRATION IN 2-D ULTRASONIC PARTICLE IMAGE VELOCIMETRY

Many recent studies on ultrasonic particle image velocimetry (Echo PIV) showed that the accuracy of two-dimensional (2-D) flow velocity measured depends largely on the concentration of ultrasound contrast agents (UCAs) during imaging. This article presents a texture-based method for identifying the optimum microbubble concentration for Echo PIV measurements in real-time. The texture features, standard deviation of gray level, and contrast, energy and homogeneity of gray level co-occurrence matrix were extracted from ultrasound contrast images of rotational and pulsatile flow (10 MHz) in vitro and in vivo mouse common carotid arterial flow (40 MHz) with UCAs at various concentrations. The results showed that, at concentration of 0.8∼2 × 10³ bubbles/mL in vitro and 1∼5 × 10⁵ bubbles/mL in vivo, image texture features had a peak value or trough value, and velocity vectors with high accuracy can be obtained. Otherwise, poor quality velocity vectors were obtained. When the texture features were used as a feature set, the accuracy of K-nearest neighbor classifier can reach 86.4% in vitro and 87.5% in vivo, respectively. The texture-based method is shown to be able to quickly identify the optimum microbubble concentration and improve the accuracy for Echo PIV imaging.

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.

Image-Guided Hydrogen Gas Delivery for Protection from Myocardial Ischemia–Reperfusion Injury via Microbubbles

Cardiomyocyte death induced by ischemia-reperfusion is a major cause of morbidity and mortality worldwide. Hydrogen (H2), as an antioxidant, has been shown to have great potential in preventive and therapeutic applications against lethal injury that occurs from ischemia-reperfusion. However, H2 is sparingly soluble in water, resulting in its poor bioavailability in blood and damaged tissues. Here, we have developed an ultrasound-visible H2 delivery system by loading H2 inside microbubbles (H2-MBs) to prevent myocardial ischemia-reperfusion injury. Using this system, the concentration of H2 in unit volume can be greatly improved under normal temperature and pressure conditions. H2-MBs can be visually tracked with ultrasound imaging systems and can effectively release their therapeutic gas. In vivo systemic delivery of H2-MBs in myocardial ischemic rats at the start of reperfusion resulted in a significant reduction of infarct size and pathological remodeling. Further analysis showed that this approach markedly inhibited cardiomyocyte apoptosis and reduced myocardial inflammation and oxidant damage in myocardial ischemia-reperfusion rats. These results indicate that H2-MBs are a promising visual delivery system for H2-based therapeutic applications.

Design and Characterization of a Tissue-Mimicking Phantom for Ultrasonic Elastography

Tissue-mimicking phantoms are essential for the investigation of ultrasonic elastography. A modified polyacrylamide gel based tissue-mimicking phantom is developed in this paper. It shows greater stability than phantom based on physical gel, such as gelatin and agar. The elastic and echographic properties of this phantom are determined by the concentration of acrylamide and graphite powder respectively. Both indentation testing and transient elastography are used to characterize the elastic properties of this phantom. The results are almost consistent with each other. Our work indicate that Youngs modulus of the polyacrylamide gel based tissue-mimicking phantom is similar to that of human soft tissue, which ranged from several kilopascal to a few tens of kilopascal.