Elsevier

Ultrasonics

Volume 44, Supplement, 22 December 2006, Pages e109-e113
Ultrasonics

Nitric oxide delivery by ultrasonic cracking: Some limitations

https://doi.org/10.1016/j.ultras.2006.06.003Get rights and content

Abstract

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 μm), 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 μm 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.

Introduction

In 1992, the radical nitric oxide (NO) was declared molecule of the year [1]. Since then, increased scientific interest has been shown in its therapeutic applications. NO is one of the 10 smallest, stable molecules of the hundreds of millions in nature [2]. The complexity of the biological processes involving NO is in contrast to the simplicity of its molecular structure [3]. In the vasculature (cf. Fig. 1), NO is produced by the endothelium and diffuses into the luminal and abluminal regions. The steady-state tissue concentration lies between 10 nM and 1 μM [4]. The average NO production by the endothelium has been estimated 6.8 × 10−14 μmol μm−2 s−1 [5]. NO traveling into smooth muscle initiates a series of reactions that lead to vessel dilation [5]. In the lumen, NO is consumed by the nearly irreversible reaction with hemoglobin within the erythrocytes. Because of this so-called scavenging, the half-life of NO in the lumen is only 1.8 ms [2], whereas the extravascular half-life of NO has been determined to be more than 90 ms [6]. The blood in the vicinity of the endothelium contains little or no erythrocytes. This thin plasma layer has been estimated between 2.6% and 12.5% of the lumen diameter [7]. NO molecules in this layer are not scavenged by erythrocytes. The consumption rate of NO has been noted to be lower in smaller vessels [8], although the plasma layers in smaller vessels are relatively thinner.

In clinical cardiology, NO finds applications in post-myocardial infarction treatment. Targeting NO to areas of early atherosclerosis might prove useful in preventing plaque formation [9]. Due to the high diffusivity of NO, however, the drug has to be applied locally or in large quantities, in order to have the effect desired. Here, ultrasound-induced bubble-assisted drug delivery may prove to be fruitful. Small quantities of NO might be administered, and released at the region of interest by means of high-amplitude ultrasound. This technique has been referred to as (ultra)sonic cracking [10].

Other microbubble-based delivery methods proposed involve mixing microbubbles with therapeutic agents [11], attaching a drug or gene to the shell [12], incorporating an oil layer inside the shell with a drug dissolved in it [13], or including therapeutic agents in antibubbles [14].

To test if the quantities of released gas are high enough to allow for NO-delivery in small vessels (ø < 200 μm), we analyzed high-speed optical and acoustical recordings of stiff-shelled microbubbles.

Section snippets

Theory

Ultrasound contrast agents have been applied in clinical diagnostics, mostly for perfusion imaging. They consist of gas microbubbles encapsulated by an elastic shell. The shell withholds the gas from dissolving, at least until it has reached a target area. Because the shell has to be biodegradable, albumins and lipids are its preferred compounds. Especially microbubbles with a stiff shell demonstrate oscillation amplitudes much lower than those of free gas bubbles of the same size. Therefore,

Methods

To illustrate the rapid dissolution of NO at 0 and 100 mmHg overpressure, Eq. (1) was solved numerically, using a Matlab® (The MathWorks, Inc., Natick, MA) program. The parameters used have been summarized in Table 1.

For the observations of ultrasonic cracking, we made use of fast framing camera systems, taking two-dimensional frames at 3 MHz and up during ultrasonic insonification. Ultrasound contrast agent microbubbles, freely flowing through a ø < 200 μm cellulose artificial vessel, were

Results and discussion

An overview of our optical observations of gas release from contrast agents has been separately presented in [19]. For both insonifying frequencies, the released gas microbubbles from PB127 have a mean diameter of 1.5 μm [19]. The mean quantity released from a single microbubble is therefore 1.7 fmol. This is already more than the average NO production of a 1 mm long vessel with a 50 μm diameter during 100 ms. A limiting factor is the low cracking rate. At high MI, less than 40% of the microbubbles

Conclusions

In clinical cardiology, NO finds applications in post-myocardial infarction treatment. Here, ultrasound-induced bubble-assisted NO delivery may prove to be helpful. Small quantities of NO might be administered, and released at the region of interest by means of high-amplitude ultrasound. Released gas may be targeted by means of primary radiation forces.

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

Acknowledgements

The authors are grateful for the contrast agent PB127 supplied by POINT Biomedical Corporation, San Carlos, CA, and for the contrast agent Quantison™ supplied by Upperton Limited, Nottingham, UK.

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