Ultrasound-mediated gene delivery: Kinetics of plasmid internalization and gene expression

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Ultrasound-mediated gene delivery: Kinetics of plasmid internalization and gene expression Sophie Mehier-Humberta,b, Thierry Bettingerb, Feng Yanb, Richard H. Guya,c,T a

b

University of Geneva, School of Pharmacy and Biopharmacy, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Bracco Research SA, Department of Novel Agents Research, 31 route de la Galaise, CH-1228 Plan-les-Ouates, Switzerland c University of Bath, Department of Pharmacy and Pharmacology, Claverton Down, Bath, BA2 7AY, England Received 24 August 2004; accepted 20 January 2005 Available online 2 March 2005

Abstract Sonoporation is an approach that can be used to transfer DNA or drugs into cells. However, very little is known about the mechanism of ultrasound-mediated membrane permeabilization. In this investigation, DNA transport post-sonoporation and the subsequent plasmid internalization and protein expression kinetics have been studied. Using a plasmid encoding for the green fluorescent protein (GFP), labelled or not with an intercalating agent (YOYO-1), it was found that, as compared to lipofection that requires endocytosis, sonoporation allowed a rapid and direct transfer of naked DNA into the cell cytoplasm probably via ultrasound-induced pores in the membrane. The kinetics of protein expression were significantly faster for sonoporation than for lipofection, the mechanism of which requires endocytosis. However, unprotected DNA in the cytoplasm could be degraded by resident cytosolic DNases, thereby decreasing ultrasound-mediated gene delivery efficiency. D 2005 Elsevier B.V. All rights reserved. Keywords: Gene delivery; Sonoporation; Mechanism; Microbubbles; Lipofection

1. Introduction One factor critical to successful human gene therapy is the development of efficient delivery systems. Although advances in gene transfer technology, including viral and non-viral vectors, have been made, with clinical trials performed and in progress, an ideal vector T Corresponding author. University of Bath, Department of Pharmacy and Pharmacology, Claverton Down, Bath, BA2 7AY, England. Tel.: +44 1225 384901; fax: +44 1225 386114. E-mail address: [email protected] (R.H. Guy). 0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2005.01.011

system has not yet been constructed. Viral vectors are very efficient but they are immunogenic [1] and more toxic than non-viral vectors [2]. In vivo, lipid-mediated gene delivery suffers from a lack of efficacy: drug or gene delivery is limited by the inability of these vectors to cross biological barriers, including the blood vessels and cell membranes. As a result, bphysicalQ methods, such as electroporation, have been considered [3,4]. It has been shown that cells can also be permeabilized using diagnostic and therapeutic ultrasound in a process termed sonoporation [5]. It has been suggested that

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ultrasound-mediated gene transfer is principally due to acoustic cavitation [6] which can be further enhanced in the presence of ultrasound contrast agents (UCA), such as LevovistR, AlbunexR or OptisonR both in vitro [5,7,8] and in vivo [9–11]. Similar to electrical pulses for electroporation, ultrasonic waves may increase cell membrane permeability by inducing transient holes in the membrane and facilitate thereby the transfer of DNA into cells [12,9]. The permeabilization and cell viability are mainly influenced by the cellular architecture [13] and sonoporation parameters [14–16]. Although numerous experimental studies have been performed to examine the biophysical effects of sonoporation on cells, the approach is not yet fully exploited and understood. In particular, the fundamental mechanisms of sonoporation, at the cellular level, need to be elucidated. For instance, how do cells take up DNA molecules and how fast does the gene expression take effect following sonoporation? What is its efficiency as compared to lipofection and what could be the factors limiting its potential clinical application? The present mechanistic study considers different aspects of sonoporation, in particular the plasmid internalization process and the kinetics of plasmid uptake. Their consequences on gene expression are also investigated, and the results are compared and contrasted to lipid-mediated gene transfer.

2. Materials and methods 2.1. Cell culture Rat mammary carcinoma cells (MAT B III), nonadherent cells, were incubated at 37 8C under an atmosphere of 5% CO2 in 225 cm2 tissue culture flasks containing a solution of Mac Coy’s 5A medium with Glutamax (Life Technologie, Switzerland) supplemented with 10% v/v foetal calf serum (FCS) and 1% v/v antibiotics (initial concentration: 10,000 IU/ml Penicillin, 10,000 Ag/ml Streptomycin, 25 Ag/ml Fungizone).

Panametrics (Waltham, MA, USA). In all sonoporation experiments, the following ultrasound settings were applied: peak negative pressure of 570 kPa (corresponding to a mechanical index of 0.38), duty cycle of 20% and pulse repetition frequency of 100 Hz. Indeed, optimization studies have showed that these US-parameters were consistent with good transfection rates and high cell viability (N 70%). Equally, using these settings, no degradation of the plasmid DNA delivered in these experiments (see below) was observed (data not shown). Briefly, in a 3 ml polystyrene round-bottom tube serving as an ultrasound exposure chamber, 500 Al of cell suspension, at a final concentration of 1.106 cells/ ml, were placed in FCS-free medium containing either the plasmid gWizk–GFP, purchased from Aldevron (Fargo, ND, USA), encoding for the green fluorescent protein (GFP, 10 Ag/ml) or the fluorescent marker, FITC-dextran 150S (MW = 167 kDa, 1% v/v) purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). An ultrasound contrast agent (UCA) provided by Bracco Research SA (Plan les Ouates, Switzerland), at a concentration of 25–30 particles/ cell, was added to the cell suspension. For plasmid internalization studies, the plasmid was fluorescently labelled with an intercalating nucleic acid stain YOYO-1 (Molecular Probes, Eugene, OR, USA). The DNA/YOYO-1 complex was prepared with a molar ratio of 1 dye molecule per 300 base pairs and an incubation of 30 min at room temperature in the dark. At this DNA/YOYO-1 ratio, DNA conformation is not affected by the presence of the nucleic acid stain. The sample tube was fixed on a rotating system in a water bath thermostated at 37 8C, with a distance of 7.6 cm between the transducer and the exposure chamber (focal distance). Then, MAT B III cells were insonified for 10 s with or without internalizing materials and UCA. For transfection experiments, after insonification, the cell suspension was placed into a 12-well plate completed with 2 ml of medium containing 10% v/v of FCS and 1% v/v of antibiotics. Finally, the cells were incubated at 37 8C under 5% CO2 for 24 h prior to analysis.

2.2. Ultrasound exposure 2.3. Liposomal transfection MAT B III cells were sonoporated using a rotating tube system [15] and a focused transducer with a transmitted frequency of 2.25 MHz, purchased from

For some experiments, sonoporation was compared to lipofection, using Lipofectamine 2000 (LF 2000),

purchased from Invitrogen (Carlsbag, CA, USA). The complex LF 2000/DNA, called lipoplex, was prepared with a LF 2000/DNA ratio of 2 / 1 v/w as recommended by the manufacturer. The transfection was carried out in a 3 ml polystyrene round-bottom tube in the culture medium without FCS, using similar plasmid and cell concentrations as described above. Then, the cells were transferred into 12-well plates, completed with 2 ml of the medium without FCS and incubated at 37 8C, under 5% CO2. Four to six hours after addition of LF 2000, the medium was discarded and a fresh medium containing 10% v/v of FCS and 1% v/v of antibiotics were added and the cells were placed in the incubator for 18–20 h prior to analysis. 2.4. Analysis of sonoporation and transfection efficacy Cells were analyzed by flow cytometry (FACS Calibur, Becton Dickinson AG, Switzerland). The cell suspension was placed in 5 ml polystyrene round-bottom tubes and washed twice with PBS. The pellet was then resuspended in 250–300 Al of PBS. In order to assess cell viability, propidium iodide (PI), purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany), a DNA intercalating agent generally excluded from viable cells, was added (20 Al of a 40 Ag/ml solution) prior to the flow cytometric analysis. Results are expressed as the percentage of positive cells and in fluorescence intensity, using the software program CellQuest Pro. The percentage of positive cells is expressed for the whole cell population, including dead cells. The fluorescence intensity is expressed in arbitrary units (AU). Plasmid internalization and particle uptake by cells following sonoporation were also analyzed by fluorescence microscopy (Olympus IX50). For the transfection experiments, GFP-positive cells were examined 24 h after sonoporation (except for GFP expression kinetics studies which were investigated at different times post-transfection). 2.5. Statistical analysis All experiments were performed in triplicates and repeated at least two or three times for reproducibility. All results are reported and displayed as mean F standard deviation. Tests of significance were

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performed using a one-way analysis of variance (MINITAB), with p b 0.05 considered to be statistically significant.

3. Results and discussion 3.1. Plasmid internalization Previous work from other groups [12] suggests that ultrasound-mediated gene delivery could be due to an enhancement of membrane permeability, probably by the formation of pores in the cell membrane. However, the plasmid internalization and its intracellular trafficking in sonoporation have not yet been fully elucidated. In contrast, DNA transfer into cells using cationic lipids as the delivery vector (lipofection), has been extensively described in the literature [17–20]. Here, we studied the process of DNA internalization into cells following sonoporation, and compared the uptake to that achieved via lipofection with Lipofectamine 2000 (LF 2000). We observed DNA transfer by fluorescence microscopy, using DNA labelled with the intercalating agent YOYO-1. Scanning confocal microscopy was not used, because YOYO-1 fluorescence was quickly destroyed by the laser beam. Fig. 1 shows punctuate fluorescent regions, representing YOYO-1 labelled LF 2000/DNA lipoplexes. Five minutes post-transfection, complexes can already be observed on the cell surface (Fig. 1A) and they migrate slowly to the nucleus. The fluorescent clusters suggest that lipoplexes were entrapped in large endocytic vesicles (Fig. 1B). Our results corroborate those in the literature which described the endocytic uptake of LF 2000/DNA complexes into cells. Sonoporation of cells showed a completely different pattern of intracellular distribution of fluorescently labelled-DNA. Indeed, following ultrasound exposure, the plasmid, which was not degraded by US treatment, was already homogeneously distributed throughout the cytoplasm, at relatively short times (5 min, Fig. 2). To further confirm this observation, we performed sonoporation at 4 8C, at which the endocytic process is greatly slowed down. This experiment was conducted using both FITC-dextran and DNA labelled with YOYO-1 as internalized molecules. We found that both FITCdextran and labelled plasmid were still successfully

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Fig. 1. Lipid-mediated plasmid internalization. MAT B III cells were incubated with LF 2000/(DNA/YOYO-1) lipoplexes and observed at (A) 5 min and (B) 1 h with a fluorescent microscope. The pictures correspond to the overlay of light transmission and epifluorescence images (using the analySIS software). Exposure time for the fluorescence microscopy observations: 100 ms. Obj.  100.

sonoporated into cells at 4 8C, albeit at a slightly reduced level (5–10%, data not shown). This is consistent with the fact that lowering the temperature significantly decreases membrane fluidity, which should, in turn, reduce pore formation as well [21]. These results confirm that sonoporation does not require endocytosis for internalization. Cavitation induced by bubble collapse may generate microjets or microstreaming allowing formation of pores in the plasma membrane and/or propelling plasmid molecules into the cells. Subsequently, the DNA may distribute throughout the cytoplasm (Fig. 2). It follows that plasmid internalization by sonoporation appears to be comparable to that described for electroporation [22–24]. Plasmid internalization was also quantified by flow cytometry to determine, as a function of time, the

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proportion of plasmid-containing cells. Internalization of YOYO-1 labelled DNA was studied in MAT B III cells following lipofection and sonoporation. Fig. 3 shows that about 30% of the cells were positive after sonoporation in the presence of UCA (these cells were viable because a subsequent experiment performed at 24 h showed that the percentage of YOYO-1 positive cells was not different—see below). However, the percentage of plasmid-internalizing cells with sonoporation was 2–3 times lower than that with lipofection. This difference may be explained as follows. First, naked DNA used in sonoporation is highly negatively charged and exists in a sterically unfavourable stretched conformation, which makes it difficult for the molecule to cross a negatively charged cell surface. In contrast, lipoplexes, which are generally positively charged, attach well to the plasma

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Fig. 2. Ultrasound-mediated plasmid internalization. MAT B III cells were sonoporated with YOYO-1 labelled DNA and observed at (A) 5 min and (B) 1 h post-sonoporation, using a focused transducer (2.25 MHz) at P- = 570 kPa in the presence of an UCA at a concentration of 25 particles/cell (using the analySIS software). Exposure time for the fluorescence microscopy observations: 500 ms. Obj.  100. Note that all the observed cells showed similar patterns of fluorescence.

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Fig. 3. Kinetics of plasmid internalization. MAT B III cells were sonoporated alone (- -x- -) or in the presence of an UCA at a concentration of 30 particles/cell (-D-), and were analyzed by flow cytometry for 24 h. For comparison, MAT B III cells were also transfected with Lipofectamine 2000 (- -5- -).

membrane. Second, not all bubble collapse induced cell membrane permeation; only that which produced sufficient cavitation allowed DNA to enter into cells. Third, as the bubble collapse was a fast process under ultrasound exposure (74% of the bubbles were destroyed within 10 s using the focused 2.25 MHz transducer, data not shown), the time available for naked DNA to enter the cell through the cavitationinduced pores was very short (the pore opening was transient with a lifetime of milliseconds to seconds, manuscript in submission). In lipofection, the internalization of DNA by cells was not limited by this factor; all DNA–lipoplexes attached at the cell surface should eventually end up inside the cell via endocytosis. The results obtained by flow cytometry (Fig. 3) show that DNA uptake appeared fast both for lipofection and sonoporation. In the case of sonoporation, these results are consistent with the fluorescence microscopy observations (Fig. 2). Plasmid DNA present in the vicinity of sonoporated cells was probably directly transferred into cells. These results confirm that ultrasound-mediated plasmid delivery is indeed an binstantaneousQ process. Surprisingly, a small increase in the percentage of DNA/YOYO-1 positive cells was observed in Fig. 3 at short times (5 min vs. 4 h postsonoporation). This could be due to a lower cell viability when cells were analyzed immediately after exposition to ultrasound. Cells were weakened by the sonoporation process and were consequently less resistant to centrifugation (cells were washed twice in PBS before flow cytometric analysis). In other words, cells need time to recover and 30% of cells internalizing plasmid is probably an under-estimation.

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It should be noted that while Taniyama et al. described poration of the cell membrane that lasted almost 24 h [9], we have found that efficient gene/drug delivery can be achieved with a duration of pore opening of less than 5 s [32]. The uptake of DNA/YOYO-1 was significantly (1.6-fold) lower without UCA. The presence of UCA might allow a more homogeneous distribution of the acoustic energy, allowing poration of more cells. The apparently rapid plasmid uptake via lipofection (~75% of positive cells within a few minutes) was mainly due to a rapid adsorption of lipoplex particles onto the cell surface. The fluorescence microscopy images in Fig. 1 support this conclusion. Since flow cytometry cannot distinguish the fluorescence emitted from the cell surface from that within the cell, the high percentage of positive cells (~90%) observed with lipofection does not necessarily mean that all these cells contained internalized DNA complexes. Zabner et al. demonstrated that the process of cationic lipid–DNA complex entry into cells was relatively slow and that most of the complexes were not internalized before 6 h [25]. It is likely that the washing procedure used in our experiments was insufficient to remove membrane-bound lipoplexes, resulting in the high internalization rates observed by flow cytometry at short times. In order to remove the LF 2000/labelled DNA bound to the cell surface, several washing protocols, using surfactant or acid were performed, with limited success and high cell toxicity. While the process of DNA attachment onto the cell membrane is rapid in lipofection, the trafficking of plasmid DNA to cell nucleus is generally slow [20,26] compared to that of sonoporation (see below). The images in Fig. 2 suggest that, in sonoporation, DNA molecules were homogeneously distributed throughout the entire cytoplasm; potentially allowing faster intracellular transfer of plasmid to the nucleus. In lipofection, the DNA complexes, once endocytosed, have to be processed in endosomes and lysosomes before delivery to the cell nucleus [20,26]. The percentage of DNA/YOYO-1 positive cells was higher than that of transfected cells (GFP-positive cells, Fig. 4), suggesting that plasmid transfer from the cytoplasm to the nucleus may involve a limiting step for DNA transfection in sonoporation. The lower values observed for GFP-positive cells could be explained by the instability of naked DNA in the

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The kinetics of gene expression from 30 min to 48 h, following sonoporation and lipofection, were assessed by flow cytometry. Fig. 6A shows that after ultrasound exposure, cells rapidly expressed GFP, with the maximum level of expression reached between 5 and 6 h. For lipofection, the gene expression started at 5–6 h, and the maximum gene expression was observed only at 24 h following plasmid incubation. Generally, the diffusion of plasmid in the cytoplasm is very slow, especially for DNA molecules larger than 2000 base pairs. For instance, Lukacs et al. showed in HeLa cells that only very few DNA molecules were able to diffuse away from the microinjection site one hour after delivery into the cytoplasm [27]. As soon as DNA enters the nucleus, protein expression takes place rapidly, within 3 h. However the expression is delayed if the DNA is introduced into the cytoplasm instead of cell nucleus because of its poor mobility [29]. The rapid kinetics of GFP observed in Fig. 5 for sonoporation could be explained by a rapid intracellular transfer (or distribution) of DNA molecules in cytoplasm; naked DNA may be bpropelledQ into the perinuclear region following sonoporation, thus facilitating nuclear uptake. The relatively slow kinetics of gene expression observed in lipofection was mainly due to the more complicated DNA delivery process via endocytosis. Lipofection is a multi-step event involving the endosomal uptake of lipoplex, the maturation of

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Fig. 4. Comparison between plasmid internalization and gene expression at 24 h post-sonoporation. Percentage of PI-positive cells (x) and percentage of plasmid-internalizing cells (DNA/ YOYO-1 positive cells) or transfected cells (GFP-positive cells) were analyzed by flow cytometry 24 h post-sonoporation at P- = 570 kPa using a focused transducer operating at a transmitted frequency of 2.25 MHz.

cytoplasm (presence of DNases) and/or slow diffusion of DNA within the cell [27]. The hypothesis that cytoplasmic DNases metabolize the internalized DNA can be verified by comparing kinetics of plasmid and FITC-dextran internalization. Fig. 5 shows a decrease in the percentage of plasmid internalizing cells over time (for t N 5 h) contrasted to the percentage of FITCdextran positive cells, which remained constant. These results suggest that DNA plasmid could be partially degraded in cytoplasm. It has been shown that YOYO-1 presents a very high affinity for doublestranded DNA [28]. However, one cannot rule out that YOYO-1 could stain other nucleic acid in the cytoplasm such as RNA, since YOYO-1 complexes are formed by electrostatic interaction. For sonoporation, protection of the naked plasmid would be required to maximize the amount eventually delivered to the cell nucleus. This hypothesis is supported by the work of Ludtke et al. who demonstrated that a 4.8 kpb plasmid, at a concentration similar to that used in our experiments (10 Ag/ml), encoding for GFP and microinjected into the cytoplasm, led to a lower transfection rate than when directly injected into the nucleus, resulting respectively in 31% and 96% of GFP-positive cells [29]. According to these observations, the results obtained in our sonoporation experiments are encouraging, since most of the plasmidinternalizing cells were transfected after sonoporation (~20% of cells, Fig. 4).

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Time (h) Fig. 5. Kinetics of DNA/YOYO-1 (-x-) and FITC-dextran 150S (- -5- -) internalization into MAT B III cells. Cells were sonoporated in the presence of an UCA at a concentration of 30 particles/ cell, using a focused transducer operating at 2.25 MHz at P- = 570 kPa, and were analyzed by flow cytometry.

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Fig. 6. Kinetics of gene expression. Mat B III cells were analyzed by flow cytometry following sonoporation (2.25MHz, P- = 570 kPa) in the presence of an UCA at a concentration of 25 particles/cell (-x-), and following lipofection (- -5- -). Results are expressed (A) as the percentage of normalized positive cells (defined as the % of positive cells at time (t) divided by the % at 24 h when the maximum level of expression was observed), and (B) in terms of fluorescence intensity (in arbitrary units: AU).

endosomes into lysosomes, escape from vesicular compartments, the migration of the DNA–lipid complex toward the nucleus periphery, the dissociation of DNA from the lipid and finally entrance of DNA into the nucleus [25]. This mechanism implies slow intracellular trafficking of DNA before gene expression can occur. Thus, compared to sonoporation, lipofection requires much more time to deliver DNA to the nucleus. These results are in good agreement with the fluorescence microscopy observations shown in Figs. 1 and 2. Fig. 6A shows a decrease of GFP-positive cells at 48 h post-transfection for both techniques. This could be due to the half-life of GFP which is on the order of one day [30]. The decrease could also be explained by cell growth, thereby reducing the quantity of GFP copies per cell. It is interesting to note, however, that reduced cell growth was observed during the 24 h following sonoporation, suggesting that cell metabolism could be affected by ultrasound exposure. Indeed, it has been shown that ultrasound affects intracytoplasmic integrity by disassembly of microtubules and microfilaments [31]. Alternatively, the apparent decrease in cell growth could be due to the fact that cellular debris was not taken into account. Although this debris probably represents only a small fraction of sonoporated cells (less than 5%), it will have contributed to an overall decrease in the total cell number. On the other hand, Fig. 6B shows that the fluorescence intensity obtained by lipofection was

7–9-fold higher than that achieved by sonoporation. Higher amounts of GFP produced by transfected cells would correspond to a higher number of DNA copies transferred into the cell nucleus. DNA condensed in lipoplexes is generally more stable in the cytoplasm toward DNases degradation than the naked DNA used with sonoporation. As a consequence, the transcription of more bintactQ DNA molecules could be achieved, resulting in a higher amount of GFP.

4. Conclusion Although ultrasound-mediated gene delivery has been studied for several years, its mechanism has not been fully elucidated. Sonoporation is a complex process, involving numerous aspects, from ultrasound bubble physics to DNA and cellular biochemistry. To design an effective gene delivery system for in vivo applications, certain fundamental aspects of sonoporation must be understood, especially those related to safety, efficacy and clinical applicability. Here, we have focused on cellular effects, specifically plasmid uptake and protein expression. Compared to lipofection, for which the internalization of DNA–lipid complexes requires endocytosis, sonoporation allowed a DNA plasmid to directly enter the cell cytoplasm, probably via the pores induced by acoustic cavitation. The results presented show that DNA plasmid and other large

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molecules (e.g., dextran of molecular weight 167 kDa) were internalized by direct transport and were distributed homogeneously throughout the whole cell cytoplasm. In contrast, complexes of LF 2000/ DNA were preferentially concentrated in endosomes or lysosomes resulting in a punctuate intracellular distribution. Unprotected, naked DNA molecules may also be degraded by DNases in the cytoplasm, thus lowering the uptake of intact DNA molecules by the nucleus for further transcription. Optimization of sonoporation will inevitably demand a compromise. The induction of more and/or larger pores in the cell membrane may allow the transfer of more materials into the cytoplasm, but such a strategy will also risk increasing cell damage, or even cell death. Whether the results presented in this study may be transposable to cells in a living organism remains to be seen. Further investigations of the mechanisms of sonoporation should address the following questions: (i) Is it possible to formulate specific ultrasound-sensitive agents (size, shell and gas) to increase bubble destruction and cavitation effect? (ii) Is DNA protection an effective approach to increase gene expression? (iii) Is there a size limit on molecules that can enter cells during sonoporation? (iv) What can be done to facilitate the transfer of DNA into cells and to cell nucleus? and (v) Can sonoporation increase the porosity/ permeability of all cell types? In terms of potential in vivo applications, it appears that sonoporation could be a useful technique to deliver DNA bkillerQ genes to tumors where cell damage is less of a concern and for which non-viral vectors are inefficient. Further, because sonoporation is a very efficient way to rapidly deliver molecules into the cytoplasm, it may prove to be an attractive and general technique for intracellular drug delivery.

Acknowledgments We thank Michel Schneider, Eric Alle´ mann, Sibylle Pochon and Jacques Terrettaz (Bracco Research SA, Geneva, Switzerland) for their helpful comments. We also thank Marcel Arditi and Peter Frinking (Bracco Research SA, Geneva, Switzerland) for their contribution to optimization of ultrasound settings.

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