Institute of Fundamental Technological Research

The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5 × 10^5 BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

Gene delivery, Sonoporation, Tendon

Nitric oxide (NO) has been implicated in smooth muscle relaxation. Its use has been widespread in cardiology. Due to the effective scavenging of NO by hemoglobin, however, the drug has to be applied locally or in large quantities, to have the effect desired. We propose the use of encapsulated microbubbles that act as a vehicle to carry the gas to a region of interest. By applying a burst of high-amplitude ultrasound, the shell encapsulating the gas can be cracked. Consequently, the gas is released upon which its dissolution and diffusion begins. This process is generally referred to as (ultra)sonic cracking.
To test if the quantities of released gas are high enough to allow for NO-delivery in small vessels (ø < 200 lm), we analyzed high-speed optical recordings of insonified stiff-shelled microbubbles. These microbubbles were subjected to ultrasonic cracking using 0.5 or 1.7 MHz ultrasound with mechanical index MI > 0.6. The mean quantity released from a single microbubble is 1.7 fmol. This is already more than the NO production of a 1 mm long vessel with a 50 lm diameter during 100 ms. However, we simulated that the dissolution time of typical released NO microbubbles is equal to the half-life time of NO in whole blood due to scavenging by hemoglobin (1.8 ms), but much smaller than the extravascular half-life time of NO (>90 ms).
We conclude that ultrasonic cracking can only be a successful means for nitric oxide delivery, if the gas is released in or near the red blood cell-free plasma next to the endothelium. A complicating factor in the in vivo situation is the variation in blood pressure. Although our simulations and acoustic measurements demonstrate that the dissolution speed of free gas increases with the hydrostatic pressure, the in vitro acoustic amplitudes suggest that the number of released microbubbles decreases at higher hydrostatic pressures. This indicates that ultrasonic cracking mostly occurs during the expansion phase.

Nitric oxide, Sonic cracking

We investigated gas release from two hard- shelled ultrasound contrast agents by subjecting them to high-mechanical index (MI) ultrasound and simultaneously capturing high-speed photographs. At an insonifying frequency of 1.7 MHz, a larger percentage of contrast bubbles is seen to crack than at 0.5 MHz. Most of the released gas bubbles have equilibrium diameters between 1.25 and 1.75 m. Their disappearance was observed optically. Free gas bubbles have equilibrium diameters smaller than the bubbles from which they have been released. Coalescence may account for the long dissolution times acoustically observed and published in previous studies. After sonic cracking, the cracked bubbles stay acoustically active.

Sonic cracking

This paper describes a noninvasive method to measure local hydrostatic pressures in fluid filled cavities. The method is based on the disappearance time of a gas bubble, as the disappearance time is related to the hydrostatic pressure. When a bubble shrinks, its response to ultrasound changes. From this response, the disappearance time, and with it the hydrostatic pressure, can be determined.
We investigated the applicability of the gases Ar, C3F8, Kr, N2, Ne, and SF6, based on their diffusive properties. For pressure measurements with a limited duration, e.g. 150 ms, Kr and Ar bubbles are most suitable, since they are most sensitive to pressure change. If there is also a limitation to bubble size, e.g. a maximum diameter of 6 lm, SF6 is most suitable.
We present improvements of a method that correlates the duration of the decay of the fundamental ultrasound response to the hydrostatic overpressure. We propose to correlate the duration until subharmonic occurrence in combination with its decay, to hydrostatic overpressure, since the subharmonic decays more rapidly than the fundamental response. For a dissolving Ar gas bubble with an initial diameter of 14 lm, the overpressure can be determined 4 times as precise from the decay of the subharmonic response as from the decay of the fundamental response. Overpressures as small as 11 mmHg may be discriminated with this method.

Noninvasive pressure measurement, Blood pressure, Microbubble, Sonic cracking

Ultrasound contrast agents (UCAs) are used in a clinical setting to enhance the backscattered signal from the blood pool to estimate perfusion and blood flow. The UCAs consist of encapsulated microbubbles, measuring 1–10 m in diameter. Acoustic characterization of UCAs is generally carried out from an ensemble of bubbles. The measured signal is a complicated summation of all signals from the individual microbubbles. Hence, characterization of a single bubble from acoustic measurements is complex.
In this study, 583 optical observations of freely flowing, oscillating, individual microbubbles from an experimental UCA were analyzed. The excursions during ultra- sound exposure were observed through a microscope. Images were recorded with a high frame rate camera operating at 3 MHz. Microbubbles on these images were measured off-line, and maximal excursions were determined. A technique is described to determine the diameters of the bubbles observed. We compared the maximal excursions of microbubbles of the same initial size in an ultrasound field with a 500 kHz center frequency at acoustic amplitudes ranging from 0.06 MPa to 0.85 MPa.
It was concluded that maximal excursions of identical bubbles can differ by 150% at low acoustic pressures (mechanical index or MI 0.2). At a high acoustic pressure (MI = 1.2) an image sequence was recorded on which a bubble collapsed, but an apparently identical bubble survived.

The cover page shows a sequence of microscopic image frames of a freely flowing contrast agent microbubble. The frames were taken during one cycle of ultrasound insonification, with a center frequency of 500 kHz. The peak negative acoustic pressure at the region of interest was 0.85 MPa. Each frame corresponds to a 45 x 27 μm2 area. The exposure time of each frame was 10 ns. Interframe times were 330 ns, except for the time between frames e and f, which was 660 ns. The sequence shows a growing gas encapsulated microbubble of 5.3 μm (a) and 17.6 μm (b), and its maximal growth of 22.9 μm (c). After shrinking to 20.2 μm (d), it ruptured (e). The microbubble had been pushed to the lower left side of the frame, apparently by water that was propelled into the microbubble. A subframe shows the negative of the region of interest. Finally, the deformed mcrobubble re-occurred as an assymetric shape (f). Understanding of microbubble-rupturing behavior is neccessary for developments in medical release burst imaging and ultra- sound-guided drug delivery. This work has been supported by the Technology Foundation STW (RKG.5104) and the Interuniversity Cardiology Institute of The Netherlands.

Ultrasound that is routinely used for imaging is now exploited for therapeutic applications including drug delivery or gene transfer. Today, ultrasound imaging is an established and confident technique for diagnosis. It is mainly based on the development of contrast imaging methods that aim to identify and display the echo from contrast agent as well as rejecting the echo from surrounding tissue offering thus a more resolutive detection. Ultrasound contrast agents or microbubbles (MB) are small gas bubbles encapsulated by a stabilizing shell, with a typical diameter of micron range. Ultrasound pulses are typically applied with a frequency near the resonance frequency of the gas bubble and the bubbles oscillations produce strong echoes from regions of perfused tissue [1-2]. Activation of microbubbles (MB) under specific ultrasound (US) beams induces a transient cell membrane permeabilization with a process known as sonoporation [3-4]. This work aims at evaluating the use of ultrasound and microbubbles for gene transfer in Achilles tendons.

Ultrasound, Sonoporation, Gene transfer

Understanding the mechanisms of microbubble destruction is needed for the development of ultrasound guided drug and gene delivery methods and for the improvement of diagnostic ultrasonic contrast agent (UCA) detection methods. We performed 482 experiments on the coalescence and collapse mechanisms of a soft- shelled and a hard-shelled contrast agent, by subjecting an experimental lipid-shelled UCA and the hard-shelled UCA QuantisonTM to 500 kHz, high- pressured ultrasound (MI≈1.0), and recording microscopic images of these events with a fast- framing camera. Results showed that bubble fragmentation into smaller bubbles is the primary mechanism for lipid-shelled contrast microbubble destruction during the first cycles after ultrasound arrival. In 28% of our experimental events with a lipid-shelled UCA, we observed bubble coalescence. The coalescence mechanism was observed to be analog to the process desribed for larger gas bubbles. Repetitive coalescence and fragmentation was clearly recorded with a fast-framing camera. We also demonstrated the formation and collapse of large lipid-shelled bubbles and bubble clusters. Furthermore we showed that sonic cracking is feasible for the hard-shelled contrast agent QuantisonTM.

In this study we analyze the behavior of individual experimental ultrasonic contrast bubbles, insonofied by 500 kHz ultrasound, at acoustic pressures between 0.06 and 0.66 MPa. The oscillations were observed under a microscope with a fast framing camera.
It is concluded that apparently identical bubbles can expand to different maximal diameters.