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Cellular membranes are, in general, impermeable to macromolecules (herein referred to as macrodrugs, e.g., recombinant protein, expression plasmids, or mRNA), which is a major barrier for clinical translation of macrodrug-based therapies. Encapsulation of macromolecules in lipid nanoparticles (LNPs) can protect the therapeutic agent during transport through the body and facilitate the intracellular delivery via a fusion-based pathway. Furthermore, designing LNPs responsive to stimuli can make their delivery more localized, thus limiting the side effects. However, the principles and criteria for designing such nanoparticles remain unclear. We show that the thermodynamic state of the lipid membrane of the nanoparticle is a key design principle for acoustically responsive fusogenic nanoparticles. We have optimized a cationic LNP (designated LNPLH) with two different phase transitions near physiological conditions for delivering mRNA. A bicistronic mRNA encoding a single domain intracellular antibody fragment and green fluorescent protein (GFP) was introduced into a range of human cancer cell types using LNPLH, and the protein expression was measured via fluorescence corresponding to the GFP expression. The LNPLH/mRNA complex demonstrated low toxicity and high delivery, which was significantly enhanced when the transfection occurred in the presence of acoustic shock waves. The results suggest that the thermodynamic state of LNPs provides an important criterion for stimulus responsive fusogenic nanoparticles to deliver macrodrugs to the inside of cells.

Shock waves are used clinically for breaking kidney stones and treating musculoskeletal indications. The mechanisms by which shock waves interact with tissue are still not well understood. Here, ultra-high-speed imaging was used to visualize the deformation of individual cells embedded in a tissue-mimicking phantom when subject to shock-wave exposure from a clinical source. Three kidney epithelial cell lines were considered to represent normal healthy (human renal epithelial), cancer (CAKI-2), and virus-transformed (HK-2) cells. The experimental results showed that during the compressive phase of the shock waves, there was a small (<2%) decrease in the projected cell area, but during the tensile phase, there was a relatively large (∼10%) increase in the projected cell area. The experimental observations were captured by a numerical model with a constitutive material framework consisting of an equation of state for the volumetric response and hyper-viscoelasticity for the deviatoric response. To model the volumetric cell response, it was necessary to change from a higher bulk modulus during the compression to a lower bulk modulus during the tensile shock loading. It was discovered that cancer cells showed a smaller deformation but faster response to the shock-wave tensile phase compared to their noncancerous counterparts. Cell viability experiments, however, showed that cancer cells suffered more damage than other cell types. These data suggest that the cell response to shock waves is specific to the type of cell and waveforms that could be tailored to an application. For example, the model predicts that a shock wave with a tensile stress of 4.59 MPa would increase cell membrane permeability for cancer cells with minimal impact on normal cells.

Microbubbles have been used in ultrasound-assisted drug delivery to help target solid tumors via blood vessels in vivo; however, studies to understand the phenomena at the cellular level and to optimize parameters for ultrasound or microbubbles in vivo are challenging and expensive to perform. Here, we utilize microfluidic microvessels-on-a-chip that enable visualization of microbubble/ultrasound-dependent drug delivery to microvasculature. When exposed to pulsed ultrasound, microbubbles perfused through microvessels-on-a-chip were observed to stably oscillate. Minimal cellular damage was observed for both microbubbles and untargeted doxorubicin-encapsulating liposomes (DOX-liposomes) perfused through chip microvessels. In contrast, passive and ultrasound-assisted perfusion of integrin-targeted DOX-liposomes induced cytotoxicity, which was only significantly enhanced for ultrasound-assisted perfusion when microbubbles were coperfused. These results suggest that stably oscillating microbubbles enhance targeted DOX-liposome internalization/cytotoxicity largely by stimulating integrin receptor endocytosis. Furthermore, our study demonstrates the utility of our microvessels-on-a-chip as a screening platform for optimizing drug dosage, targeting ligands and drugs.

Background and Purpose: During SWL, stone motion secondary to ventilatory motion can be as much as 50 mm. This is much larger than the 4- to 15-mm diameter of the focal regions on most clinical machines. The goal of this study was to determine the effect of stone motion on the fragmentation efficiency of a clinical lithotripter with a small (4-mm) focal spot.Materials and Methods: A model stone (6.5 × 7.5 mm) made of gypsum cement was used as an in vitro target for a Storz Modulith SLX lithotripter with a custom-designed waterbath. A motorized positioner was used to translate the stone in order to simulate ventilatory motion. The excursion was variable up to 48 mm (± 24 mm about the focus). After treatment by 400 shockwaves, the remnants (>2 mm) were dried and weighed.Results: Fragmentation efficiency was reduced (P<0.05) for motion of ≥10 mm. Similar results were found with different energy levels and firing rates. The reduction in fragmentation efficiency was consistent with calculations of the time the stone was outside the focal region.Conclusions: Clinically relevant stone motion has a dramatic effect on in vitro comminution. Motion of 10 mm led to a significant reduction in comminution, and for motion >20 mm, it appeared that three-quarters of the shockwaves missed the stone. These data imply that ventilatory gating or stone tracking may result in fewer shockwaves being required for successful treatment with this lithotripter.

Background and Purpose: There is strong evidence that cavitation bubble activity contributes to stone breakage and that shockwave-bubble interactions are involved in the tissue trauma associated with shockwave lithotripsy. Cavitation control may thus be a way to improve lithotripsy.Materials and Methods: High-speed photography was used to analyze cavitation bubble activity at the surface of artificial and natural kidney stones during exposure to lithotripter shockwaves in vitro.Results: Numerous individual bubbles formed on the surfaces of stones, but these bubbles did not remain independent but rather combined to form clusters. Bubble clusters formed at the proximal and distal ends and at the sides of stones. Each cluster collapsed to a narrow point of impact. Collapse of the proximal cluster eroded the leading face of the stone, and the collapse of clusters at the sides of stones appeared to contribute to the growth of cracks. Collapse of the distal cluster caused minimal damage.Conclusion: Cavitation-mediated damage to stones is attributable, not to the action of solitary bubbles, but to the growth and collapse of bubble clusters.

Using human whole blood samples with and without contrast agent (CA), we evaluated the effect of exposures to focused, continuous wave (CW) 1.1-MHz ultrasound for durations of 10 ms to 1 s at spatial average intensities of 560 to 2360 W/cm2. Cavitation was monitored with a passive cavitation detector and hemolysis was determined with spectroscopy. In whole blood alone, no significant cavitation, heating or hemolysis was detected at any exposure condition. Conversely, cavitation and hemolysis, but not heating, were detected in whole blood with CA. A CA concentration as low as 0.28 μL CA per mL whole blood at an intensity of 2360 W/cm2for 1 s resulted in measurable cavitation and a 6-fold increase in hemolysis compared to shams. Cavitation and hemolysis increased proportional to the concentration of CA and duration of exposure. In samples containing 4.2 μL CA per mL whole blood exposed for 1 s, a threshold was seen at 1750 W/cm2where cavitation and hemolysis increased 10-fold compared to exposures at lower intensities. HIFU exposure of whole blood containing CA leads to significant hemolysis in vitroand may lead to clinically significant hemolysis in vivo.

Increased ambient pressure (excess hydrostatic pressure) was used to regulate cavitation in the aqueous media surrounding isolated red blood cells and aluminum foils used as targets in studies of the mechanisms of shock wave (SW) damage in shock wave lithotripsy (SWL). Foils or cells were placed in a cylindrical chamber (bronze-aluminum alloy) connected to a regulator and nitrogen source. The ends of the cylinder were capped with planar, plastic plates 12.7 mm thick. Tests performed with a PVDF membrane hydrophone showed that SW's passed through the plastic without significant loss of amplitude or change in waveform. Pitting of foils, a form of damage that can only be due to cavitation, was eliminated by very high (∼70 atm) added pressure. It took substantially greater added pressure to reduce damage to foils than has been reported to reduce damage to cells, suggesting differences in how cavitation is regulated in bulk fluid versus a solid surface. This report describes the design of the pressure chamber used in these studies and explains how SW exposures of isolated cells and foil targets were performed.

A numerical exercise to compare computer codes for the propagation of sonic booms through idealized atmospheres is reported. Ground waveforms are calculated using four different codes or algorithms: (1) weak shock theory, an analytical prediction; (2) SHOCKN, a mixed time and frequency domain code developed at the University of Mississippi; (3) ZEPHYRUS, another mixed time and frequency code developed at the University of Texas; and (4) THOR, a pure time domain code recently developed at the University of Texas. The codes are described and their differences noted. They are then used to calculate propagation for two different source waveforms, for both a uniform and isothermal (varying density) atmosphere, with and without the presence of molecular relaxation. In all cases the results of THOR, SHOCKN, and ZEPHYRUS are in excellent agreement. Because the weak shock theory algorithm does not include the effect of ordinary absorption, it does not predict the shock structure provided by the other codes. This leads to discrepancies particularly for an atmosphere that includes relaxation effects. © 1996 Acoustical Society of America.

A time‐domain computer algorithm that solves an augmented Burgers equation is described. The algorithm is a modification of the time‐domain code developed by Lee and Hamilton [J. Acoust. Soc. Am. 97, 906–917 (1995)] for pulsed finite‐amplitude sound beams in homogeneous, thermoviscous fluids. In the present paper, effects of nonlinearity, absorption and dispersion (both thermoviscous and relaxational), geometrical spreading, and inhomogeneity of the medium are taken into account. The novel feature of the code is that effects of absorption and dispersion due to multiple relaxation phenomena are included with calculations performed exclusively in the time domain. Numerical results are compared with an analytic solution for a plane step shock in a monorelaxing fluid, and with frequency‐domain calculations for a plane harmonic wave in a thermoviscous, monorelaxing fluid. The algorithm is also used to solve an augmented KZK equation that accounts for nonlinearity, thermoviscous absorption, relaxation, and diffraction in directive sound beams. Calculations are presented which demonstrate the effect of relaxation on the propagation of a pulsed, diffracting, finite‐amplitude sound beam. © 1996 Acoustical Society of America.

Polypropylene vials are commonly used in invitro experiments to hold cell preparations that are exposed to ultrasound. The vial has an acoustic impedance very close to water, in which case there should be little transmission loss as sound propagates into the vial. Measurements of the acoustic field within polypropylene vials have been carried out using both pulsed medical ultrasound and lithotripter shock waves. It has been found that for certain orientations of the acoustic field and vial there is significant loss of pressure amplitude within the vial. In particular, sound that enters the vial through the round, hemispherical end is attenuated and distorted. Hot and cold spots within the vial are also measured. When the rounded end is replaced with a flat surface very little distortion and attenuation of the waveform occurs. The distortion induced by the round end is apparently due to refraction introduced by the vial—the speed of sound in polypropylene is about 1.7 times that of water. A simple ray analysis indicates the presence of hot and cold spots within the vial in qualitative agreement with observations. These results indicate that cell injury experiments may be dependent on the vial orientation. [Work supported by NIH and the ASA Hunt Fellowship.]

This laboratory is involved in studies to determine the mechanism(s) responsible for cellular injury to the kidney during shock wave lithotripsy for the treatment of upper urinary tract stones. The studies are performed using an electrohydraulic lithotripter (Dornier HM3) and injury to isolated, cultured renal tubular epithelial cells was assessed. In this demonstration our experiences with the practical aspects of performing invitro SW exposures are shared. Issues to be discussed and/or demonstrated include the following: (a) choice of cultured cell models and the handling of isolated cells, (b) factors that influence invitro cell injury, (c) selection and preparation of specimen vials, (d) lithotripter setup and operation, (e) positioning the specimen and control of exposure parameters, (f) electrode performance and variability, (g) characterization of waveforms and determination of SW pressures, (h) markers of cell injury and cell response to SW insult, and (i) safety in the lithotripsy suite. [Work supported by NIH Program Project Grant No. PO1 43881: Role of SWL in renal injury and stone comminution.]

Blast exposure is associated with chronic traumatic encephalopathy, impaired neuronal function, and persistent cognitive deficits in blast-exposed military veterans and experimental animals.

The rise time of a sonic boom shock in air depends strongly on the relaxation processes of nitrogen and oxygen. Stratification of the atmosphere leads to significant variation of the relaxation processes with altitude. J. Kang [Ph.D. thesis, Penn State (1991)] argues that the shock profile can adjust quickly enough to changes in attenuation that it always appears to be in steady state. If correct, then rise time at the ground can be calculated from local conditions only. A time domain computer algorithm, based on work by Lee and Hamilton (‘‘Time domain modeling of pulsed finite‐amplitude sound beams,’’ submitted to J. Acoust. Soc. Am. in March 1994), is presented for a Burgers‐type equation. The algorithm includes the effects of nonlinear distortion, thermoviscous absorption, molecular relaxation, and ray tube spreading. A parametric study of the effect of change in relative humidity shows that the steady‐state assumption is not justified for sonic boom shocks in the atmosphere. A sonic boom propagated through a real atmosphere is shown to have a rise time that, in all cases run so far, is shorter than that of a steady‐state shock calculated using atmospheric properties at the ground only. [Work supported by NASA.]

Ellipsoidal reflectors are widely used in electrohydraulic lithotripters. The small‐amplitude, continuous wave (cw) reflected field of a small spherical source that is placed at the first focus (f1) of an ellipsoidal reflector is investigated with both theory and experiment. This configuration is equivalent to a low‐amplitude (linear regime), cw‐driven lithotripter. To model the acoustic field reflected from an ellipsoidal reflector, geometrical acoustics is used to define a directivity function at the mouth of the reflector. An equivalent focused source with amplitude shading defined by the directivity function is then assumed as the boundary condition for the linear, lossless parabolic wave equation which is used to diffractively propagate the field outside the reflector. The spherical source is driven with a long tone burst at 470 kHz. The ellipsoidal reflector is made out of nickel and has a rhodium coating. The major and minor radii are 6.2 cm and 3.5 cm, respectively. Good agreement is found between experiment and theory. A useful property of ellipsoidal reflectors is that one can quickly and accurately position the source at f1 and the hydrophone at f2 by a simple iteration technique. [Work supported by NIH.]

The propagation of intense waves in liquids or gases with relaxation processes is a classical problem of nonlinear acoustics. The presence of relaxing processes influences acoustic propagation by changing the absorption law and introducing dispersion. The effect of the relaxation is of particular interest for short acoustic pulses, when the duration of pulse is of the same order as the characteristic relaxation time. Even for the case of linear acoustics, relaxation will compromise pulse shape. For intense waves, nonlinear effects result in additional pulse distortion, shock formation, and nonlinear energy attenuation. The propagation of intense acoustic pulses containing shocks is studied theoretically and experimentally. Two numerical simulations, a modified spectral method and a time‐domain method, are used to model shock propagation. Propagation curves for waveform distortion, shock amplitude behavior, and energy dissipation are presented. Experiments are performed in acetic acid as a monorelaxing medium. Short acoustic pulses are excited by a pulsed laser. Theoretical and experimental results are in a good agreement. [Work supported by NIH, FIRCA, and RFBR.]