Introduction to Microbubbles and Their Use in Sonoporation

Microbubbles have been used for many years as contrast agents for use in diagnostic ultrasound. More recently, however, they have been shown to be very effective for use in the targeted delivery of drugs and genes (1-6).

The mechanism of intracellular delivery using ultrasound is believed to be initiated by local fluid dynamics generated by oscillating and/or collapsing cavitation bubbles (7). By either shearing or direct impact of fluid with the cell its plasma membrane can be torn open to produce a hole of up to 1μm in size (8,9). Molecules can transport through this hole to gain access to the cytosol. Cells, in turn, traffic intracellular vesicles to the site of injury and actively repair the hole on a timescale of a few minutes. The presence of microbubbles has been shown to lower the amount of ultrasound required to have the desired effect.

There are several different ways in which microbubbles designed for use with ultrasound have been modified to incorporate drugs for delivery. The drugs have been incorporated into the microbubble of the shell, and site-specific ligands have been attached to the outside of the shell to allow tissue specific targeting. These bubbles are filled with perfluorocarbon. They are stable and capable of circulating in the blood system until the target area is reached. When ultrasound is applied over the area of interest it can burst the microbubbles leading to localized release of the drug (10-13) with the result that less of the toxic drug is required to elicit the desired effect due to the localized delivery.

There are several factors that contribute to the effectiveness of the microbubble ultrasound-enhanced uptake: the interplay of the therapeutic agent, the microbubble characteristics, the target tissue, and the nature of the ultrasound. The microbubbles in the presence of ultrasound act as cavitation nuclei, which decreases the threshold for ultrasound energy to create the pores in the cell membrane.

In 2002 Taniyama, et al [10] demonstrated the presence of small holes in the surface of endothelial and vascular smooth muscle cells immediately after transfection of a plasmid DNA by ultrasound-mediated microbubble destruction, using electron microscopic scanning. It was then postulated that these transient holes in the cell surface caused by microbubbles and ultrasound resulted in a rapid translocation of plasmid DNA from outside to cytoplasm.

In 2000, Mukherjee, et al [13] used electron microscopy to examine the effects on a rat heart during ultrasound application. They showed that disruption or pore formation in the membrane of endothelial cells occurred when acoustic powers of 0.8-1W/cm 2 to 1W/cm were used. It was also shown that a lower intensity of ultrasound at 0.6W/cm was more effective for drug uptake.

The first published report of targeted DNA delivery was performed in 1996, using surface ultrasound and intravenously delivered microbubbles carrying antisense oligonucleotides [6].

In 1997, Bao, et al [26] described the use of ultrasound and albumin-coated microbubbles to enhance the transfection of luciferase reporter plasmid in cultured hamster cells. Since then, many studies have confirmed the efficacy of ultrasound-mediated microbubble destruction for drug and gene delivery, both in vitro and in vivo [6,10-12].

Taniyama, et al [10] published on the effective transfection of a plasmid DNA to endothelial and vascular smooth muscle cells with albumin-coated microbubbles (Optison) and ultrasound. The in-vivo studies demonstrated that transfection of wild-type p53 plasmid DNA into balloon-injured blood vessels was effective and resulted in significant inhibition of the ratio of neointimal-to-medial area, as compared with transfection of control vector. By contrast, when cells were transfected with p53 DNA using ultrasound alone no microbubbles, the cells failed to inhibit Neointimal formation in the rat carotid [10].

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2. Price RJ, Skyba DM, KAUL S, Skalak TC: Delivery of colloidal particles and red blood cells to tissue through microvessel ruptures created by targeted microbubble destruction with ultrasound. Circulation 1998, 98: 1264-1267.

3. Porter TR, Iversen PL, Li S, Xie F: Interaction of diagnostic ultrasound with synthetic oligonucleotide labeled perfluorocarbon- exposed sonicated dextrose albumin microbubbles. J Ultrasound Med 1996, 15: 577-584.

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6. Unger EC, McCreery TP, Sweitzer RH, Caldwell VE, Wu Y: Acoustically active lipospheres containing paclitaxel: a new therapeutic ultrasound contrast agent. Invest Radiol 1998, 33: 886-892.

7. Prentice, P., Cuschierp, A., Dholakia, K., Prausnitz, M. R., Campbell, P. Membrane disruption by optically controlled microbubble cavitation. Nature Physics 1 , 107-110 (2005).

8. McNeil, P. L, Steinhardt, R. A. Plasma membrane disruption: repair, prevention, adaptation. Annu Rev Cell Dev Biol 19 , 697-731 (2003).

9. Schlicher, R. K., Radkhakrishna, H., Tolentino, T. P., Apkarian, R. P., Zarnitsyn, V., Prausnitz, M. R. Mechanism of intracellular deivery by acoustic cavitation. Ultrasound Med Biol 32 , 915-924 (2006).

10. Taniyama Y, Tachibana K, Hiraoka K, Namba T, Yamasaki K, HashiyavN, et al. : Local delivery of plasmid DNA into rat carotid artery v using ultrasound. Circulation 2002, 105: 1233-1239.

11. Chen S, Shohet RV, Bekeredjian R, Frenkel P, Grayburn PA: Optimization of ultrasound parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by ultrasound targeted microbubble destruction. J Am Coll ardiol 2003, 42: 301-308.

12. Shohet RV, Chen S, Zhou YT, Wang Z, Meidell RS, Unger RH, et al. : Echocardiographic destruction of albumin microbubbles

directs gene delivery to the myocardium. Circulation 2000, 101: 2554-2556.

13. Mukherjee D, Wong J, Griffin B, Ellis SG, Porter T, Sen S, et al. : Tenfold augmentation of endothelial uptake of vascular endothelial growth factor with ultrasound after systemic administration. J Am Coll Cardiol 2000, 35: 1678-1686.

14. Villanueva FS, Jankowski RJ, Manaugh C, Wagner WR: Albumin

microbubble adherence to human coronary endothelium: implications for assessment of endothelial function using myocardial contrast echocardiography. J Am Coll Cardiol 1997, 30: 689-693.