Juan Tu

The correlation between acoustic cavitation and sonoporation involved in ultrasound-mediated DNA transfection with polyethylenimine (PEI) in vitro

Previous studies have demonstrated that the efficiency of gene/drug delivery can be enhanced under ultrasound (US) exposure with the presence of US contrast agent microbubbles, due to the acoustic cavitation-induced sonoporation. However, obstacles still remain to achieve controllable sonoporation outcome. The general hypotheses guiding present studies were that inertial cavitation (IC) activities accumulated during US exposure could be quantified as IC dose (ICD) based on passive cavitation detection (PCD), and the assessment of sonoporation outcome should be correlated with ICD measurements. In current work, MCF-7 cells mixed with PEI:DNA complex and UCD microbubbles were exposed to 1-MHz US pulses with 20-cycle pulse and varied acoustic peak negative pressure (P(-); 0 (sham), 0.3, 0.75, 1.4, 2.2 or 3.0MPa), total treatment time (0, 5, 10, 20, 40 or 60s), and pulse-repetition-frequency (PRF; 0, 20, 100, 250, 500, or 1000Hz). Then, four series experiments were conducted: (1) the IC activities were detected using a PCD system and quantified as ICD; (2) the DNA transfection efficiency was evaluated with flow cytometry; (3) the cell viability was examined by PI dying then measured using flow cytometry; and (4) scan electron microscopy was used to investigate the sonoporation effects on the cell membrane. The results showed that: (1) the ICD generated during US exposure could be affected by US parameters (e.g., P(-), total treatment time, and PRF); (2) the pooled data analyses demonstrated that DNA transfection efficiency initially increased linearly with the increasing ICD, then it tended to saturate instead of trying to achieve a maximum value while the ICD kept going up; and (3) the measured ICD, sonoporation pore size, and cell viability exhibited high correlation among each other. All the results indicated that IC activity should play an important role in the US-mediated DNA transfection through sonoporation, and ICD could be used as an effective tool to monitor and control the US-mediated gene/drug delivery effect.

Estimating the shell parameters of SonoVue ® microbubbles using light scattering

Experiments were performed to measure the dynamical response of individual SonoVue microbubbles subjected to pulsed ultrasound. Three commonly used bubble dynamic models (i.e., Hoffs, Sarkars, and linearized Marmottants models) were compared to determine the most appropriate model for fitting to the experimental data. The models were evaluated against published optical microscopy data. The comparison suggests that it is difficult to rank these models for lipid-shelled microbubbles undergoing small-amplitude oscillations, because under these conditions the shell parameters in these models are closely related. A linearized version of the Marmottant model was used to estimate the shell parameters (i.e., shear modulus and shear viscosity) of SonoVue microbubbles from the experimental light scattering data, as a function of ambient microbubble radius. The SonoVue microbubble shell elasticity and dilatational viscosity increase with ambient bubble radius, in agreement with previously published data. The results suggest that light scattering, used in conjunction with one of several popular bubble dynamics models, is effective at characterizing microbubble response and evaluating shell parameters.

Microbubble-induced sonoporation involved in ultrasound-mediated DNA transfection in vitro at low acoustic pressures.

In the present work, human breast cancer cells MCF-7 mixed with polyethylenimine: deoxyribonucleic acid complex and microbubbles were exposed to 1-MHz ultrasound at low acoustic driving pressures ranging from 0.05 to 0.3 MPa. The sonoporation pores generated on the cell membrane were examined with scanning electron microscopy. The transfection efficiency and cell viability were evaluated with flow cytometry. The results showed that ultrasound sonication under the current exposure condition could generate cell pores with mean size ranging from about 100 nm to 1.25 μm, and that larger sonoporation pores would be generated with the increasing acoustic pressure or longer treatment time, leading to the enhancement of transfection efficiency and the reduction of cell viability. The simulations based on the Marmottant model were performed to test the hypothesis that the microstreaming-induced shear stress might be involved in the mechanisms of the low-intensity ultrasound induced sonoporation. The calculated shear stress resulting from the micro-streaming ranged from 15 to 680 Pa corresponding to the applied acoustic pressures 0.05-0.3 MPa, which is sufficient to induce reversible sonoporation. This study indicates that the shear stress related bio-effects may provide a base for strategies aimed at targeted drug delivery.

Dispersionless Manipulation of Reflected Acoustic Wavefront by Subwavelength Corrugated Surface

Free controls of optic/acoustic waves for bending, focusing or steering the energy of wavefronts are highly desirable in many practical scenarios. However, the dispersive nature of the existing metamaterials/metasurfaces for wavefront manipulation necessarily results in limited bandwidth. Here, we propose the concept of dispersionless wavefront manipulation and report a theoretical, numerical and experimental work on the design of a reflective surface capable of controlling the acoustic wavefront arbitrarily without bandwidth limitation. Analytical analysis predicts the possibility to completely eliminate the frequency dependence with a specific gradient surface which can be implemented by designing a subwavelength corrugated surface. Experimental and numerical results, well consistent with the theoretical predictions, have validated the proposed scheme by demonstrating a distinct phenomenon of extraordinary acoustic reflection within an ultra-broad band. For acquiring a deeper insight into the underlying physics, a simple physical model is developed which helps to interpret this extraordinary phenomenon and predict the upper cutoff frequency precisely. Generations of planar focusing and non-diffractive beam have also been exemplified. With the dispersionless wave-steering capability and deep discrete resolution, our designed structure may open new avenue to fully steer classical waves and offer design possibilities for broadband optical/acoustical devices.

Microbubble sizing and shell characterization using flow cytometry

Experiments were performed to size, count, and obtain shell parameters for individual ultrasound contrast microbubbles using a modified flow cytometer. Light scattering was modeled using Mie theory, and applied to calibration beads to calibrate the system. The size distribution and population were measured directly from the flow cytometer. The shell parameters (shear modulus and shear viscosity) were quantified at different acoustic pressures (from 95 to 333 kPa) by fitting microbubble response data to a bubble dynamics model. The size distribution of the contrast agent microbubbles is consistent with manufacturer specifications. The shell shear viscosity increases with increasing equilibrium microbubble size, and decreases with increasing shear rate. The observed trends are independent of driving pressure amplitude. The shell elasticity does not vary with microbubble size. The results suggest that a modified flow cytometer can be an effective tool to characterize the physical properties of microbubbles, including size distribution, population, and shell parameters.

Unidirectional acoustic transmission based on source pattern reconstruction

It is demonstrated both theoretically and experimentally that unidirectional acoustic transmission can be achieved through a simple structure consisting of only a uniform stiff plate and periodic rectangular gratings. Essentially distinct from the previous related works based on the utilizing of nonlinear effect or the partial band of the sonic crystal, it is attributed to one-side pattern reconstruction of acoustic plane waves induced by the periodic gratings, which can be physically modeled by forced vibration of the plate under periodic loads. The current structure takes the advantages of high transmission efficiency, small size, and broad bandwidth, which should open more possibilities for the miniaturization and integration of the one-way acoustic devices as well as further improvement of their performance.

Evaluation of a shock wave induced cavitation activity both in vitro and in vivo

This study evaluated the cavitation activity induced by shock wave (SW) pulses, both in vitro and in vivo, based on the area measurements of echogenic regions observed in B-mode ultrasound images. Residual cavitation bubble clouds induced by SW pulses were detected as echogenic regions in B-mode images. The temporal evolution of residual bubble clouds, generated by SWs with varying lithotripter charging voltage and pulse repetition frequency (PRF), was analyzed by measuring the time-varying behaviors of the echogenic region areas recorded in B-mode images. The results showed that (1) the area of SW-induced echogenic regions enlarged with increased SW pulse number; (2) echogenic regions in the B-mode images dissipated gradually after ceasing the SWs, which indicated the dissolution of the cavitation bubbles; and (3) larger echogenic regions were generated with higher charging voltage or PRF.

Acoustic non-diffracting Airy beam

The acoustic non-diffracting Airy beam as its optical counterpart has unique features of self-bending and self-healing. The complexity of most current designs handicaps its applications. A simple design of an acoustic source capable of generating multi-frequency and broad-band acoustic Airy beam has been theoretically demonstrated by numerical simulations. In the design, a piston transducer is corrugated to induce spatial phase variation for transducing the Airy function. The pistons surface is grooved in a pattern that the width of each groove corresponds to the half wavelength of Airy function. The resulted frequency characteristics and its dependence on the size of the piston source are also discussed. This simple design may promote the wide applications of acoustic Airy beam particularly in the field of medical ultrasound.

Ultrasound-enhanced protective effect of tetramethylpyrazine against cerebral ischemia/reperfusion injury.

In traditional Chinese medicine, Ligusticum wallichii (Chuan Xiong) and its bioactive ingredient, tetramethylpyrazine (TMP), have been used to treat cardiovascular diseases and to relieve various neurological symptoms, such as those associated with ischemic injury. In the present study, we investigated whether ultrasound (US) exposure could enhance the protective effect of TMP against cerebral ischemia/reperfusion (I/R) injury. Glutamate-induced toxicity to pheochromocytoma (PC12) cells was used to model I/R injury. TMP was paired with US to examine whether this combination could alleviate glutamate-induced cytotoxicity. The administration of TMP effectively protected cells against glutamate-induced apoptosis, which could be further enhanced by US-mediated sonoporation. The anti-apoptotic effect of TMP was associated with the inhibition of oxidative stress and a change in the levels of apoptosis-related proteins, Bcl-2 and Bax. Furthermore, TMP reduced the expression of proinflammatory cytokines such as TNF-α and IL-8, which likely also contributes to its cytoprotective effects. Taken together, our findings suggest that ultrasound-enhanced TMP treatment might be a promising therapeutic strategy for ischemic stroke. Further study is required to optimize ultrasound treatment parameters.

Investigation on the inertial cavitation threshold and shell properties of commercialized ultrasound contrast agent microbubbles

The inertial cavitation (IC) activity of ultrasound contrast agents (UCAs) plays an important role in the development and improvement of ultrasound diagnostic and therapeutic applications. However, various diagnostic and therapeutic applications have different requirements for IC characteristics. Here through IC dose quantifications based on passive cavitation detection, IC thresholds were measured for two commercialized UCAs, albumin-shelled KangRun(®) and lipid-shelled SonoVue(®) microbubbles, at varied UCA volume concentrations (viz., 0.125 and 0.25 vol. %) and acoustic pulse lengths (viz., 5, 10, 20, 50, and 100 cycles). Shell elastic and viscous coefficients of UCAs were estimated by fitting measured acoustic attenuation spectra with Sarkars model. The influences of sonication condition (viz., acoustic pulse length) and UCA shell properties on IC threshold were discussed based on numerical simulations. Both experimental measurements and numerical simulations indicate that IC thresholds of UCAs decrease with increasing UCA volume concentration and acoustic pulse length. The shell interfacial tension and dilatational viscosity estimated for SonoVue (0.7 ± 0.11 N/m, 6.5 ± 1.01 × 10(-8) kg/s) are smaller than those of KangRun (1.05 ± 0.18 N/m, 1.66 ± 0.38 × 10(-7) kg/s); this might result in lower IC threshold for SonoVue. The current results will be helpful for selecting and utilizing commercialized UCAs for specific clinical applications, while minimizing undesired IC-induced bioeffects.