Ultrasound and microbubble-targeted delivery of therapeutic compounds: ICIN Report Project 49: Drug and gene delivery through ultrasound and microbubbles

Abstract

The molecular understanding of diseases has been accelerated in recent years, producing many new potential therapeutic targets. A noninvasive delivery system that can target specific anatomical sites would be a great boost for many therapies, particularly those based on manipulation of gene expression. The use of microbubbles controlled by ultrasound as a method for delivery of drugs or genes to specific tissues is promising. It has been shown by our group and others that ultrasound increases cell membrane permeability and enhances uptake of drugs and genes. One of the important mechanisms is that microbubbles act to focus ultrasound energy by lowering the threshold for ultrasound bioeffects. Therefore, clear understanding of the bioeffects and mechanisms underlying the membrane permeability in the presence of microbubbles and ultrasound is of paramount importance. (Neth Heart J 2009;17:82-6.).

Keywords: bioeffects; cell membrane permeability; local therapy; microbubbles; ultrasound.

Figure 1

Experimental set-up. Ultrasound transducer (a) is mounted on the live-cell fluorescence microscope (b) to study the effects of ultrasound-exposed microbubbles in detail at the cellular level. The transducer is connected to an arbitrary wave-form generator (c) and a linear 60-dB power amplifier (e). The ultrasound signal was monitored by a synchronised oscilloscope (d).

Figure 2

Calcium influx and hyperpolarisation. Fluorescent images from a time-lapse recording. (A, B) Cells loaded with Fluo4, a green fluorescent probe sensitive for free cytosolic calcium. (C, D) Cell loaded with Di-4-ANEPPS, a red fluorescent probe sensitive for changes in membrane potential. An increase in fluorescence corresponds to hyperpolarisation of the cell membrane (indicated by arrows). (A, C) Levels of fluorescence before ultrasound is switched on. (B, D) Increased levels of fluorescence during ultrasound exposure.

Figure 3

Schematic overview of unravelled bioeffects and mechanisms. Ultrasound and microbubbles induced generation of H2O2 (1). There was a causal relationship between H2O2 and the formation of transient pores in the cell membrane with a concomitant calcium influx (2). The calcium ions activated the large-conductance potassium channels, thereby causing local hyperpolarisation of the cell membrane (3). Besides formation of transient pores, ultrasound and microbubbles induced uptake of macromolecules (dextran 500 kDa) via endocytosis (4). Ultrasound and microbubbles further affected ROS homeostasis, and caused a decrease in total gluthation (GSx) levels (5). Other unravelled effects of ultrasound and microbubbles were rearrangement and increased number of F-actin stress cables (6), and disruption of cell-cell interactions (7).

Figure 4

Deformation of the cell membrane by an insonified microbubble. A sequence of 12 images (a selection out of a 128-image sequence) is shown, in which the first cycle of 1 MHz ultrasound applied on a Sonovue™ microbubble close to an endothelial cell is observed. The interaction between the oscillating microbubble and the cell results in cell deformation. In the right panel the deformation in relation to the oscillating microbubble is depicted. Under the rarefaction effect of ultrasound, the microbubble expands to a maximum of 15 μm, whereby it presses against the cell. This maximal expansion is translated to a displacement of the cell membrane up to 2.3 μm (~15%). The compression of the microbubble is also translated into a displacement of the cell membrane. Thus, microbubble vibrations are translated into pushing and pulling of the cell membrane.