Characterization of ultrasound-mediated destruction of drug-loaded microbubbles using an improved in vitro model

Introduction

Ultrasound mediated destruction of microbubbles (MBs) has become a promising tool for site specific drug and gene delivery. One of the most important properties of drug-loaded MBs is their destructibility by ultrasound. Therefore, the aim of this study was to establish a new in vitro model that allows evaluation of the kinetics of ultrasound-mediated MB destruction at near physiological conditions.In this work, a newly developed drug-loaded MB formulation was compared with unloaded MBs in order to assess the influence of drug-loading on their acoustic destructibility. Furthermore, drug-loaded MBs were compared to acoustically active lipospheres (AALs), comprising an additional layer of triacetin, as well as to a marketed MB contrast agent (SonoVue^®, Bracco Diagnostics, USA), used as standard.

Methods

MBs with phospholipid monolayer shells were produced by mechanical agitation of liposomal dispersions and octafluoropropane gas. AALs were accordingly produced by agitation of phospholipid-stabilized aqueous triacetin microemulsions with gas.The in vitro experimental setup for acoustic destructibility testing comprised a membrane cell, pressurized and brought to 37 °C in order to imitate human blood pressure and body temperature. The optimized egg-like cell shape provided optimal flow conditions and a minimized dead volume.Ultrasound with frequencies of 1 and 3 MHz and intensities, varying from 1 to 4 W/cm², was applied through a silicone membrane window to the cell. MB size distribution and concentration were measured by light blockage in equal time intervals during the sonication.

Results

The optimized in vitro setup demonstrated differences in the ultrasound destructibility of the MB formulations used. The fastest decay upon ultrasound exposure was found for SonoVue^®. Unloaded and drug-loaded MBs appeared to be comparably destructible to SonoVue^®. AALs were about 4.5-fold more stable than SonoVue^®. MB destructibility was also found to depend on particle diameter, corresponding to theoretical models described in the literature.

Conclusion

The optimized in vitro setup has rendered a fast and reliable laboratory tool for characterization of MB formulations.