Microbubbles in ultrasound-triggered drug and gene delivery

The ability of collapsing (cavitating) bubbles to focus and concentrate energy, forces and stresses is at the root of phenomena such as cavitation damage, sonochemistry or sonoluminescence1, 2. In a biomedical context, ultrasound-driven microbubbles have been used to enhance contrast in ultrasonic images3. The observation of bubble-enhanced sonoporation4, 5, 6?acoustically induced rupture of membranes?has also opened up intriguing possibilities for the therapeutic application of sonoporation as an alternative to cell-wall permeation techniques such as electroporation7 and particle guns8. However, these pioneering experiments have not been able to pinpoint the mechanism by which the violently collapsing bubble opens pores or larger holes in membranes. Here we present an experiment in which gentle (linear) bubble oscillations are sufficient to achieve rupture of lipid membranes. In this regime, the bubble dynamics and the ensuing sonoporation can be accurately controlled. The use of microbubbles as focusing agents makes acoustics on the micrometre scale (microacoustics) a viable tool, with possible applications in cell manipulation and cell-wall permeation as well as in microfluidic devices.

/pdf/controlled-vesicle-deformation-and-lysis-by-single-3j24gqiy7b.pdf

Controlled vesicle deformation and lysis by single oscillating bubbles

Bubbles in liquids, soft and squeezy objects made of gas and vapour, yet so strong as to destroy any material and so mysterious as at times turning into tiny light bulbs, are the topic of the present report. Bubbles respond to pressure forces and reveal their full potential when periodically driven by sound waves. The basic equations for nonlinear bubble oscillation in sound fields are given, together with a survey of typical solutions. A bubble in a liquid can be considered as a representative example from nonlinear dynamical systems theory with its resonances, multiple attractors with their basins, bifurcations to chaos and not yet fully describable behaviour due to infinite complexity. Three stability conditions are treated for stable trapping of bubbles in standing sound fields: positional, spherical and diffusional stability. Chemical reactions may become important in that respect, when reacting gases fill the bubble, but the chemistry of bubbles is just touched upon and is beyond the scope of the present report. Bubble collapse, the runaway shrinking of a bubble, is presented in its current state of knowledge. Pressures and temperatures that are reached at this occasion are discussed, as well as the light emission in the form of short flashes. Aspherical bubble collapse, as for instance enforced by boundaries nearby, mitigates most of the phenomena encountered in spherical collapse, but introduces a new effect: jet formation, the self-piercing of a bubble with a high velocity liquid jet. Examples of this phenomenon are given from light induced bubbles. Two oscillating bubbles attract or repel each other, depending on their oscillations and their distance. Upon approaching, attraction may change to repulsion and vice versa. When being close, they also shoot self-piercing jets at each other. Systems of bubbles are treated as they appear after shock wave passage through a liquid and with their branched filaments that they attain in standing sound fields. The N-bubble problem is formulated in the spirit of the n-body problem of astrophysics, but with more complicated interaction forces. Simulations are compared with three-dimensional bubble dynamics obtained by stereoscopic high speed digital videography.

Physics of bubble oscillations

This selective review of the biological effects of ultrasound presents a synopsis of our current understanding of how cells insonated in vitro are affected by inertial cavitation from the standpoint of physical and chemical mechanisms. The focus of this review is on the physical and chemical mechanisms of action of inertial cavitation which appear to be effective in causing biological effects. There are several fundamental conditions which must be satisfied before cavitation-related bioeffects may arise. First, bubbles must be created and then brought into proximity to cells. Exposure methods are critical in this regard, and simple procedures such as rotation of a vessel containing the cells during exposure can drastically alter the results. Second, once association is achieved between bubbles and cells, the former must interact with the latter to produce a bioeffect. It is not certain that the inertial event is the prime mechanism by which cells are lysed; there is evidence that the turbulence associated with bubble translation may cause lysis. Additionally, there appear to be chemical and other physical mechanisms by which inertial cavitation may affect cells; these include the generation of biologically effective sonochemicals and the apparent emission of ultraviolet (UV) and soft X-rays. The evidence for inertial cavitation occurring within cells is critically reviewed.

A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective

https://ieeexplore.ieee.org/iel5/10282/32715/01534718.pdf

Acoustic Cavitation

Human (A431 epidermoid carcinoma) cells were grown as monolayers on 5 microm thick Mylar sheets, which formed the upper window for a 1-mm thick, 23-mm diameter disc-shaped exposure chamber. A 3.5-MHz curved linear-array transducer was aimed upward at the chamber, 7 cm away, in a 37 degrees C water bath. The chamber contained phosphate-buffered saline (PBS) with 10 mg/mL fluorescent dextran and 1% Optison ultrasound (US) contrast agent. Significant fluorescent cell counts, indicative of membrane damage (i.e., sonoporation), up to about 10% of cells within a 1-mm diameter field of view, were noted for spectral Doppler and two-dimensional (2-D) scan mode with or without a tissue-mimicking phantom. The effect was only weakly dependent on pulse-repetition frequency or exposure duration, but was strongly dependent on contrast agent concentration below 2%. Thus, diagnostic US activation of contrast-agent gas bodies can produce cell membrane damage.

Sonoporation of monolayer cells by diagnostic ultrasound activation of contrast-agent gas bodies.

Human platelets were induced by 2.1-megahertz ultrasound to form aggregates around gas-filled pores in membranes immersed in platelet-rich plasma. The spatial peak intensities required were only about 16 to 32 milliwatts per square centimeter. Ultrasound generated by a medical Doppler device, whose intensity exceeded this, induced aggregate formation under the same conditions.

https://science.sciencemag.org/content/sci/205/4405/505.full.pdf

Platelet aggregation induced by ultrasound under specialized conditions in vitro

Murine spleen cell suspensions stimulated by Concanavalin A (Con-A) were exposed to 1.6-MHz continuous-wave ultrasound at low intensities (spatial-peak values ranging from 16 to 300 mW/cm2) in the presence of a Nuclepore membrane that contained stabilized gas bodies. The ultrasonically activated gas bodies induced cell lysis and reduced the fraction of intact cells that excluded trypan blue. At a spatial-peak intensity of 75 mW/cm2 (spatial-average intensity 15 mW/cm2), Con-A-induced Methyl[3H]thymidine (3H-TdR) incorporation was reduced in cultures exposed at 24 or 48 hr after addition of Con-A but not at 7 or 13 hr. There was no observable effect on cell survival or 3H-TdR incorporation at spatial-peak intensities below 75 mW/cm2.

Stable cavitation at low ultrasonic intensities induces cell death and inhibits 3H-TdR incorporation by Con-A-stimulated murine lymphocytes in vitro.

Resonant bubble detectors (RBD) were used to search for both pre-existing bubbles and bubbles created by cavitation within the cardiovascular system of 22 dogs. No pre-existing bubbles of 3.8 micron diam or larger were found, nor were any created by exposing the left ventricle to 0.51-1.61 MHz ultrasound of up to 16 W/cm2 spatial-peak intensity. Bubbles introduced into the arterial system by high speed injection were readily detected and could be held in the heart by 1 MHz ultrasound at 1-2 W/cm2 or above. A surprising observation was that gas bubbles of resonant size injected into the left ventricle and held by ultrasound did not multiply continuously as happens in saline or water in vitro. This in vivo system was designed to assess the potential for cavitation bioeffects and the essentially negative results obtained may limit the expected or potential risk of this mechanism in regard to medical applications of ultrasound.

A search for ultrasonic cavitation within the canine cardiovascular system

The hypothesis that ultrasonically induced membrane damage and cell death in Elodea leaves is caused by shear stress associated with microstreaming flow generated in the vicinity of oscillating gas-filled channels between the cells is investigated. Cell death thresholds as a function of frequency seem to follow a condition of constant shear stress, with minimum thresholds near the resonance frequency of a gas-filled channel. Theoretical estimates of the shear stress generated within the cells at the ultrasonic intensity of the cell death threshold are in order of magnitude agreement with measurements of the shear stress required for lysis of blood cells. Furthermore, the dependence of the cell death thresholds on exposure duration seems to correspond roughly with the dependence of critical shear stress for blood cells as a function of the duration of the stress. Membrane damage induced by microstreaming shear stress therefore appears to be a plausible mechanism of cell death in Elodea, and a valuable unifying concept for consideration of bioeffects of ultrasonic cavitation.

Douglas L. Miller

Papers

Sonoporation of monolayer cells by diagnostic ultrasound activation of contrast-agent gas bodies.

Platelet aggregation induced by ultrasound under specialized conditions in vitro

Stable cavitation at low ultrasonic intensities induces cell death and inhibits 3H-TdR incorporation by Con-A-stimulated murine lymphocytes in vitro.

A search for ultrasonic cavitation within the canine cardiovascular system

Microstreaming shear as a mechanism of cell death in Elodea leaves exposed to ultrasound