Abstract
Therapeutic ultrasound (TUS) has the potential of becoming a powerful nonviral method for the delivery of genes into cells and tissues. Understanding the mechanism by which TUS delivers genes, its bioeffects on cells and the kinetic of gene entrances to the nucleus can improve transfection efficiency and allow better control of this modality when bringing it to clinical settings. In the present study, direct evidence for the role and possible mechanism of TUS (with or without Optison) in the in vitro gene-delivery process are presented. Appling a 1 MHz TUS, at 2 W/cm2, 30%DC for 30 min was found to achieve the highest transfection level and efficiency while maintaining high cell viability (>80%). Adding Optison further increase transfection level and efficiency by 1.5 to three-fold. Confocal microscopy studies indicate that long-term TUS application localizes the DNA in cell and nucleus regardless of Optison addition. Thus, TUS significantly affects transfection efficiency and protein kinetic expression. Using innovative direct microscopy approaches: atomic force microscopy, we demonstrate that TUS exerts bioeffects, which differ from the ones obtained when Optison is used together with TUS. Our data suggest that TUS alone affect the cell membrane in a different mechanism than when Optison is used.
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References
Fechheimer M, Boylan J, Parker S, Sisken J, Patel G, Zimmer S . Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading. Proc Natl Acad Sci USA 1987; 84: 8463–8467.
Kim HJ, Greenleaf JF, Kinnick RR, Bronk JT, Bolander ME . Ultrasound mediated transfection of mammalian cells. Hum Gene Ther 1996; 7: 1339–1346.
Tata DB, Dunn F, Tindall DJ . Selective clinical ultrasound signals mediate differential gene transfer and expression in two human prostate cancer cell lines: LnCap and PC-3. Biochem Biophys Res Com 1997; 234: 64–67.
Bao S, Thrall BD, Miller DL . Transfection of a reporter plasmid into cultured cells by sonoporation in vitro. Ultrasound Med Biol 1997; 23: 953–959.
Lawrie A, Brisken AF, Francis SE, Tayler DI, Chamberlain J, Crossman DC et al. Ultrasound enhances reporter gene expression after transfection of vascular cells in vitro. Circulation 1999; 99: 2617–2620.
Miller MW, Miller DL, Brayman AA . A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective. Ultrasound Med Biol 1996; 22: 1131–1154.
Guzman HR, McNamara AJ, Nguyen DX, Prausnitz MR . Bioeffects caused by changes in acoustic cavitation bubble density and cell concentration: a unified explanation based on cell-to-bubble ratio and blast radius. Ultrasound Med Biol 2003; 29: 1211–1222.
Ng KY, Liu Y . Therapeutic ultrasound: its application in drug delivery. Med Res Rev 2002; 22: 204–223.
Miller DL, Pislaru SV, Greenleaf JE . Sonoporation: mechanical DNA delivery by ultrasonic cavitation. Somat Cell Mol Genet 2002; 27: 115–134.
Apfel RE, Holland CK . Gauging the likelihood of cavitation from short-pulls, low-duty cycle diagnostic ultrasound. Ultrasound Med Biol 1991; 17: 179–185.
Taniyama Y, Tachibana K, Hiraoka K, Aoki M, Yamamoto S, Matsumoto K et al. Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Therapy 2002; 9: 372–380.
Brayman AA, Coppage ML, Vaidya S, Miller MW . Transient poration and cell surface receptor removal from human lymphocytes in vitro by 1 MHz ultrasound. Ultrasound Med Biol 1999; 25: 999–1008.
Lawrie A, Brisken AF, Francis SE, Tayler DI, Wyllie D, Kiss-Toth E et al. Ultrasound-enhanced transgene expression in vascular cells is not dependent upon cavitation-induced free radicals. Ultrasound Med Biol 2003; 29: 1453–1461.
Dalecki D . Mechanical bioeffects of ultrasound. Annu Rev Biomed Eng 2004; 6: 229–248.
Lawrie A, Brisken AF, Francis SE, Cumberland DC, Crossman DC, Newman CM . Microbubble-enhanced ultrasound for vascular gene delivery. Gene Therapy 2000; 7: 2023–2027.
Unger EC, Hersh E, Vannan M, McCreery T . Gene delivery using ultrasound contrast agents. Echocardiography 2001; 18: 355–361.
Alonso JL, Goldmann WH . Feeling the forces: atomic force microscopy in cell biology. Life Sci 2003; 72: 2553–2560.
Munkonge FM, Dean DA, Hillery E, Griesenbach U, Alton EW . Emerging significance of plasmid DNA nuclear import in gene therapy. Adv Drug Del Rev 2003; 55: 749–760.
Zelphati O, Liang X, Hobart P, Felgner PL . Gene chemistry: functionally and conformationally intact fluorescent plasmid DNA. Hum Gene Ther 1999; 10: 15–24.
Mehier-Humbert S, Bettinger T, Yan F, Guy RH . Plasma membrane poration induced by ultrasound exposure: Implication for drug delivery. J Control Rel 2005; 104: 213–222.
Tachibana K, Uchida T, Ogawa K, Yamashita N, Tamura K . Induction of cell-membrane porosity by ultrasound. Lancet 1999; 353: 1409.
Cochran SA, Prausnitz MR . Sonoluminescence as an indicator of cell membrane disruption by acoustic cavitation. Ultrasound Med Biol 2001; 27: 841–850.
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.
Skyba DM, Price RJ, Linka AZ, Skalak TC, Kaul S . Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue. Circulation 1998; 98: 290–293.
Taniyama Y, Tachibana K, Hiraoka K, Namba T, Yamasaki K, Hashiya N et al. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation 2002; 105: 1233–1239.
Acknowledgements
This work was in part supported by The Israel Science Foundation (ISF) to Marcelle Machluf.
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Supplementary Information accompanies the paper on the Gene Therapy website (http://www.nature.com/gt).
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Duvshani-Eshet, M., Baruch, L., Kesselman, E. et al. Therapeutic ultrasound-mediated DNA to cell and nucleus: bioeffects revealed by confocal and atomic force microscopy. Gene Ther 13, 163–172 (2006). https://doi.org/10.1038/sj.gt.3302642
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DOI: https://doi.org/10.1038/sj.gt.3302642
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