2013 http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, Early Online: 1–8 ! 2013 Informa UK Ltd. DOI: 10.3109/1061186X.2012.761223 RESEARCH ARTICLE Bleomycin delivery into cancer cells in vitro with ultrasound and SonoVue Õ or BR14 Õ microbubbles N. Lamanauskas 1,2 , A. Novell 2 , J.-M. Escoffre 2 , M. Venslauskas 1 , S. S ˇ atkauskas 1 , and A. Bouakaz 2 1 Department of Biology, Faculty of Natural Sciences, Vytautas Magnus University, Kaunas, Lithuania and 2 UMR Imagerie et Cerveau, Inserm U 930, Universite ´ Franc ¸ois Rabelais, Tours, France Abstract Background: Cell exposure to ultrasound (US) in the presence of contrast agent microbubbles (MBs) can result in cell sonoporation that can be exploited for drug or gene delivery. Anticancer drug bleomycin (BLM), used in sonoporation, can effectively eliminate tumor cells in vitro and in vivo. Nevertheless, sonoporation mechanism is not known, thus different US parameters and MB types are used. Recently, we proposed that efficiency of cell sonoporation can be related to the efficiency of MB sonodestruction. Purpose: We analyzed human tumor cells viability in response to BLM, US and MB treatment. Methods: Human glioblastoma astrocytoma (U-87 MG) or colon cancer (HCT-116) cells were exposed to US in the presence of BLM and either SonoVue Õ or BR14 Õ MBs. MB sonodestruction was evaluated according to US signal attenuation. Results: Both HCT-116 and U-87 MG cell viability following US exposure decreased up to 30%. Decrease in cell viability followed similar tendency as MB sonodestruction, which suggests direct relationship between MB sonodestruction and BLM intracellular delivery. Conclusion: Sonoporation is a feasible method to deliver BLM in to several types of human cancer cell lines. Efficiency of cell sonoporation correlated well with MB sonodestruction, providing a possibility to optimize US parameters by measuring MB sonodestruction. Keywords Bleomycin, BR14, microbubble, sonodestruction, sonoporation, SonoVue History Received 5 October 2012 Revised 4 December 2012 Accepted 18 December 2012 Published online 21 January 2013 Introduction Bleomycin (BLM) is a nonpermanent, water-soluble glyco- peptidic antibiotic, cytotoxic for eukaryotic cells and used in anticancer chemotherapy [1]. It has been shown in vitro that less than 0.1% BLM, added to the extracellular medium, enters the cells via endocytosis [2,3]. Since the transport of BLM is limited by the low number of receptors that internalize it via endocytosis, BLM is relatively an ineffective drug [1,2]. Nevertheless, once in a cell, BLM possesses a very high intrinsic cytotoxicity and induces cell killing via DNA strand breaks by an oxygen- and metal ion-dependent process in mammalian cells which are seen as chromosomal gaps, deletions and DNA fragmentation, which leads to a cell death [2,4,5]. Few hundreds of BLM molecules internalized into cytoplasm are sufficient to kill the cell [6]. This has been efficiently exploited for enhancement of BLM cytotoxicity by cell electroporation [7,8] that ultimately led to development of a new antitumor treatment – electrochemotherapy [9]. Recently, ultrasound (US) and microbubbles (MBs) as contrast agents have been proposed as a drug delivery system through a process named sonoporation [10,11]. Via sonoporation, drug delivery can be directed to many parts of the organism fully non-invasively [12]. US-mediated delivery can be controlled and therefore sonoporation can be precisely localized inducing only negligible side effects [13]. It has been shown that sonoporation can facilitate intracellular delivery of various molecules, including drugs and DNA [10,12,14,15]. Few recent studies have shown that BLM, when combined with US and MB, can effectively eliminate tumor cells both in vitro and in vivo [16–20]. However, it is difficult to understand which of the US parameters are responsible for efficient drug delivery. Indeed, in these studies, US parameters such as intensity, exposure time and duty cycle varied up to 100-fold in some cases. Moreover, different types of MBs (SonoVue Õ and Optison Õ ) were used in these studies. The great variation in US parameters indicates that they are selected largely empirically. Recently, we proposed that sonoporation can be related to MB sonodestruction [21]. Thus, the aim of our study was to analyze the efficiency of BLM delivery into human tumor cells in response to MB sonodestruction. Two kinds of human tumor cells were used in order to test the method for different tumor cells. Address for correspondence: Ayache Bouakaz, UMR Imagerie et Cerveau, Inserm U930, Universite ´ Franc ¸ois Rabelais, CHU Breton- neau–B1A, 2 Bd Tonnelle ´, 37044 Tours Cedex 9, France. Tel: þ33 02 47479748. Fax: þ33 02 47479767. E-mail: ayache.bouakaz@univ- tours.fr S ˇ atkauskas, Department of Biology, Faculty of Natural Sciences, Vytautas Magnus University, Kaunas, Lithuania. E-mail: s.satkauskas@gmf.vdu.lt Journal of Drug Targeting Downloaded from informahealthcare.com by INSERM on 02/21/13 For personal use only.