(420f) Ultrasound Induced Destruction of Liposomes Using Vaporizing Emulsion Droplets

Several groups have reported the encapsulation and delivery of Doxorubicin and other drugs from liposomes.  More recently, ultrasound has been utilized as a method to release the encapsulated drugs^1-3.  This method of targeted delivery could be more useful with liposomes that are designed to be more susceptible to ultrasound without compromising stability, allowing for a faster and more complete release of drugs when the ultrasound is applied.  In this study perfluorocarbon emulsion droplets were encapsulated inside of liposomes to increase the rate and amount of liposome destruction with ultrasound.  Upon insonation, the perfluorocarbon emulsion droplets vaporize and disrupt the liposomal membrane as they expand (Figure 1).      

Emulsion droplets were formed by mixing perfluorohexane, DPPC, and water.  A 20-kHz ultrasound probe was used to shear the solution, and the resulting emulsion was extruded through a 0.1 µm filter to reduce the size of the stabilized droplets to approximately 100 nm.  Emulsion droplets were encapsulated inside of liposomes using a ?vesosome? technique described by Kisak, et al⁴.  Dipalmitoylphosphatidylcholine (DPPC) liposomes were formed by film hydration and extruded through a 0.1 µm filter.  The 100 nm liposomes were stirred while ethanol was added dropwise to a final concentration of 3M, causing them to fuse into bilayer sheets.  Ethanol was removed from the sheet solution by centrifugation and resuspension of the pellet in excess PBS.  This process was repeated 3 times.  Concentrated calcein was added to the sheet solution with or without emulsion droplets and samples were heated to 50ºC for at least 20 minutes to reform closed vesicles.  The resulting vesicles were extruded through a 0.8 µm filter.  The average size was determined to be 800 nm by dynamic light scattering.  Unencapsulated calcein was removed from the outside of the vesicles by size exclusion chromatography using a Sephadex G25 column.  A third sample was prepared by mixing an emulsion suspension and a liposome suspension, thus placing the emulsion droplets only on the outside of the liposomes.

The liposome solutions were diluted in a sample cuvette and the amount of calcein released from the liposomes was quantified using a fluorometer.  A 20-kHz ultrasonic probe was immersed directly into the sample cuvette through an opening in the fluorometer.  Excitation and emission wavelengths were set at 488 and 525 nm, respectively.  At the conclusion of each experiment, Triton X-100 was added to the cuvette to destroy the liposomes, thus recording the fluorescence level of 100% release.  Data was normalized by fractional calcein released.   Sample data is shown in Figure 2.

The presence of internal emulsion droplets increased the amount and rate of calcein release compared to conventional liposomes, particularly at low intensities.  External emulsion droplets also increased the amount and rate of calcein release from liposomes, although not to the level of encapsulated emulsion droplets.  Further work is required at varied ultrasonic parameters and with different liposomal formulations to optimize ultrasonically-mediated release from liposomes and to describe ultrasonic thresholds for vaporization of perfluorocarbon liquids.

References

(1) Ning, S.; Macleod, K.; Abra, R.; Huang, A. H.; Hahn, G. M. Int. J. Radiation Oncology Phys. 1994, 29, 827-834.

(2) Vyas, S. P.; Singh, R.; Asati, R. K. J Microencapsul 1995, 12, 149-154.

(3) Unger, E. C.; McCreery, T. P.; Sweitzer, R. H.; Caldwell, V. E.; Wu, Y. Invest Radiol 1998, 33, 886-892.

(4) Kisak, E.T., et al. Current Medicinal Chemistry, 2004. 11(2):  199-219.