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Subcellular impact of sonoporation on plant cells: Issues to be addressed in ultrasound-mediated gene transfer
Ultrasonics Sonochemistry 20 (2013) 247–253
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Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Subcellular impact of sonoporation on plant cells: Issues to be addressed in ultrasound-mediated gene transfer Peng Qin a, Lin Xu b, Ping Cai c, Yaxin Hu a, Alfred C.H. Yu a,⇑ a
Medical Engineering Program, The University of Hong Kong, Pokfulam, Hong Kong Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China c Department of Instrumentation Science and Engineering, Shanghai Jiaotong University, Shanghai, China b
a r t i c l e
i n f o
Article history: Received 29 May 2012 Received in revised form 31 July 2012 Accepted 1 August 2012 Available online 11 August 2012 Keywords: Sonoporation Tobacco BY-2 cells Plasma membrane Reactive oxygen species Cytochrome-c Programmed cell death
a b s t r a c t Sonoporation (membrane perforation via ultrasonic cavitation) is known to be realizable in plant cells on a reversible basis. However, cell viability may concomitantly be affected over the process, and limited knowledge is now available on how such cytotoxic impact comes about. This work has investigated how sonoporation may affect plant cells at a subcellular level and in turn activate programmed cell death (PCD). Tobacco BY-2 cells were used as the plant model, and sonoporation was applied through a microbubble-mediated approach with 100:1 cell-to-bubble ratio, free-field peak rarefaction pressure of either 0.4 or 0.9 MPa, and 1 MHz ultrasound frequency (administered in pulsed standing-wave mode at 10% duty cycle, 1 kHz pulse repetition frequency, and 1 min duration). Fluoroscopy results showed that sonoporated tobacco cells may undergo plasma membrane depolarization and reactive oxygen species elevation (two cellular disruption events closely connected to PCD). It was also found that the mitochondria of sonoporated tobacco cells may lose their outer membrane potential over time (observed using confocal microscopy) and consequently release stores of cytochrome-c proteins (determined by Western Blotting) into the cytoplasm to activate PCD. These findings provide insight into the underlying mechanisms responsible for sonoporation-induced cytotoxicity in plant cells. They should be taken into account when using this membrane perforation approach for gene transfection applications in plant biotechnology. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction The use of physical means to create transient pores on the plasma membrane (PM) has long been regarded as a non-viral alternative to facilitate gene delivery to biological cells [1,2]. Ultrasound is well-considered as one of the emerging physical approaches, and it generally involves the use of acoustic cavitation principles (i.e. interaction between ultrasound and gaseous cavities) to create microjet forces for PM puncturing on a non-specific and presumably reversible basis [3]. It is now accepted that ultrasound-induced membrane perforation, often referred to as sonoporation [4], may take place even in the presence of a rigid cell wall: a component that is unique to plant cells and not found in mammalian cells [5]. This can potentially benefit the plant biotechnology field, given that gene transfection is an essential step in transgenic crop development [6] and existing transformation techniques based on agrobacterium mediation, particle bombardment, or direct protoplast transformation all have known systemic limitations [7]. Nevertheless, sonoporation as a gene transfection strategy is not yet ready for routine use in plant biotechnology, with one of the main ⇑ Corresponding author. E-mail address: [email protected] (A.C.H. Yu). 1350-4177/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2012.08.008
reasons being that this way of puncturing cells is known to concomitantly affect the ensuing viability of plant cells [5,8]. To account for this issue, it is necessary to gain an in-depth understanding of how sonoporation may bring about lethal impact in plant cells and, thereby, develop strategies to counteract against these deleterious effects. For plant cells, investigations on sonoporation-induced stress response are rather limited at present, and it remains unclear as to why cells may not be able to sustain the pulsed stress brought about by sonoporation. A few early studies conducted using ultrasonic homogenizers and sonicators have provided initial evidence that ultrasound may possibly modify the plant cells’ ion pump activities [9,10] and activate their defense response [11,12]. However, it is inherently difficult to draw a direct correlation between sonoporation and the bioeffects identified in these studies because ultrasonic homogenizers tend to induce a wide range of nonlinear physical phenomena, including shock waves and cavitation [3], on an uncontrollable basis. In this regards, perhaps a more suitable way of investigating the stress response elicited by sonoporation on plant cells would be to make use of a microbubble-mediated sonoporation paradigm where synthetic microbubbles are introduced nearby cell samples to serve as cavitation nuclei and calibrated ultrasound pressure levels are used to trigger acoustic
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cavitation. Such a setup can more systematically facilitate acoustic cavitation whose extent is significantly influenced by the peak rarefaction pressure and microbubble shell properties [13]; indeed, it is already widely adopted for sonoporation studies in mammalian cells [14,15]. Using the microbubble-mediated approach to perform sonoporation, we report here new insights on how sonoporation may affect plant cells at a subcellular level and thereby upset their viability. We have been working with the hypothesis that sonoporation-induced stress response is likely due to the disruption of a chain of cellular physiology events that are known to be involved in maintaining the viability of plant cells. Identification of these cellular disruption events is strategically important because this knowledge can provide essential insights on how we can possibly mediate certain steps in the cytoplasmic signaling cascade to mitigate the attrition issue for ultrasound-based gene transfection in plant cells. Our current work will specifically focus on evaluating the post-sonoporation response of: (1) PM potential, (2) intracellular reactive oxygen species (ROS) level, (3) mitochondrial outer membrane (MOM) potential and (4) cytochrome-c (cyto-c) protein expression. As will be discussed, for these quantities, their deviations from the homeostatic level are known to lead to activation of the programmed cell death (PCD) protocol [16,17]. 2. Materials and methods 2.1. Cell culturing protocol Our investigation was performed on Nicotiana tabacum cells (cultivar Bright Yellow 2) that are well considered as a generic model for higher plant physiology studies [18]. Using the Murashige–Skoog (MS) medium as the culturing medium, the tobacco cells were grown in suspension under a 27 °C dark environment with 120 rpm agitation. The culturing medium was supplemented with sucrose (3%), mono-potassium phosphate (200 mg/L), thiamine (1 mg/L), myo-inositol (100 mg/L), and 2,4-dichlorophenoxy acetic acid (0.2 mg/L; pH 5.8); also, subculturing of cells was performed after every three days. The cells were fostered to grow in the exponential phase before they were harvested for experiments. Note that, prior to receiving sonoporation exposure, the harvested cells were washed three times with phosphate buffered saline (PBS) to remove secondary metabolic products present in the cell samples. 2.2. Sonoporation procedure Sonoporation was applied to the harvested tobacco cells using the exposure apparatus shown in Fig. 1. At the top of this apparatus
is a 15 mm-diameter well plate (176740; Nunc, Rosklide, Denmark) containing a 0.9 mL cell–microbubble mixture (cell density: 106 cells/mL; microbubble volume density: 1% v/v; height of mixture inside well: 5 mm), and ultrasound pulses were transmitted to the well plate from the unfocused transducer that is positioned at the bottom of the water bath. We have previously shown that this exposure setup may bring about sonoporation in BY-2 cells at peak rarefaction ultrasound pressures approaching the microbubble disruption threshold (i.e. when inertial cavitation becomes more significant), but in the process it may concomitantly lead to necrosis and PCD in parts of the cell population [5]. The microbubbles that serve as synthetic cavitation nuclei in this work are essentially lipid-shelled gas bodies. They were fabricated using a perfluorobutane gas sonication procedure as described earlier [19], and their shell material comprises a combination of phosphatidylcholine and polyethylene glycol stearate. Their size ranged from 1 to 5 lm, and the original density in each vial of microbubbles was 106 bubbles/mL. Thus, with a 1% v/v microbubble concentration, the cell-to-bubble ratio was about 100:1 for each experimental trial. Note that we have deliberately chosen to use this microbubble concentration because a high cell-to-bubble ratio (i.e. having more cells than microbubbles) has been previously found to be efficient in facilitating sonoporation without concomitantly inducing significant amounts of cell lysis [20]. In turn, it would allow us to focus more on analyzing the cellular disruption events inside sonoporated cells that remain intact after exposure. The ultrasound transducer used in the exposure setup is of 1-in. diameter and has a 1 MHz center frequency (P1T10W-25; Kunshan Risun Electronic Co., Jiangsu, China). It operated in a tone-burst mode with 1 kHz pulse repetition frequency and 10% duty cycle, and it was driven by a waveform generator (33120A; Agilent Technologies, Palo Alto, USA) with broadband amplification (2100L; Electronics & Innovation Ltd., Rochester, USA). Its excitation voltage was calibrated to generate free-field peak rarefaction pressures of 0.4 or 0.9 MPa at the well-plate region 70 mm away from transducer, as measured using a capsule hydrophone (HGL-0400; Onda Corporation, Sunnyvale, USA). These two pressure levels were chosen as we have previously determined that, for the microbubbles used in our work, 0.4 MPa free-field peak rarefaction pressure predominantly facilitates stable cavitation that generates non-collapsing microbubble pulsations, whilst 0.9 MPa would generate more inertial cavitation activities involving rapid expansion and collapse of microbubbles [5]. Note that, for our exposure apparatus, the actual in situ pressure may be higher since the upper surface of the well plate was coupled to a reflecting air interface that is known to contribute to the presence of standing waves within the cell–microbubble
Tobacco Cells 106 cells/mL density with 104 bubbles/mL Well Plate 70 mm
Signal Amplifier Generator Waveform N-cycle sinusoids, 10% duty cycle 1 kHz pulse repetition freq., 1 min. duration
Water Bath
Transducer Ultrasound 1 MHz frequency 0.4 or 0.9 MPa peak negative pressure
Fig. 1. Illustration of the experimental setup used for sonoporation of tobacco cells. It is based on ultrasound-microbubble mediated cavitation principles, as our team has shown previously [5].
P. Qin et al. / Ultrasonics Sonochemistry 20 (2013) 247–253
mixture [21]. Specifically, given that the height of mixture inside the well was 5 mm (equivalent to 3.25 times the acoustic wavelength), approximately 5–6 antinodal planes parallel to the air interface would emerge inside the well plate, and their pressure level may be up to twice the free-field amplitude. In turn, the actual amount of sonoporation activity may be enhanced as a result [22]. It should be emphasized that the acoustic pressure levels used in this study, even for those at the standing-wave antinodal planes, are well-considered as being low such that other nonlinear acoustic phenomena like shock waves and heating would not be inflicted [23]. As well, acoustic cavitation would not be generated at these pressure levels unless synthetic microbubbles are present to serve as artificial cavitation nuclei [24]. Correspondingly, it can be inferred that the bioeffects observed in our study are generally a result of sonoporation generated by ultrasound-microbubble mediated cavitation. We have confirmed this in our preliminary experiments where no subcellular bioeffects were detected in the tobacco cells after ultrasound exposure if synthetic microbubbles were not introduced into the medium (data not shown). 2.3. Monitoring of PM potential and ROS level After the end of exposure, the sonoporated tobacco cells were re-incubated under pre-exposure culturing conditions. To characterize the post-exposure level of PM potential and intracellular ROS, samples in each exposure group were extracted at four different time points (0, 2, 4, and 6 h). These two quantities were respectively labeled in situ using two different fluorescence dyes. PM potential was labeled with the DiBAC4(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] voltage-dependent fluorescence dye (ENZ-52205; Enzo Life Sciences, Plymouth, USA). It was carried out by adding 0.25 lM DiBAC4(3) [buffered in a 2 mM dimethyl sulphoxide (DMSO) stock] to each sonoporated cell population sample (100 lL in volume) that was extracted through PBS washing at 200 g for 5 min. For intracellular ROS, it was tracked with the H2DCF-DA (20 ,70 -dichlorofluorescencein diacetate) fluorescence dye (D-399; Molecular Probes, Eugene, USA). Similar to the DiBAC4(3) labeling procedure, 3 lM of the H2DCF-DA dye was directly added to each sample. Each cell population sample labeled with DiBAC4(3) or H2DCFDA was allowed to further incubate for at least 30 min in a dark, room-temperature (RT) environment with gentle agitation. It was then washed with PBS, and the resulting sample was observed under a differential interference contrast (DIC) microscope (DS-Ri1; Nikon Instruments, Melville, USA). DiBAC4(3) fluorescence was observed using excitation and detection wavelengths of 493 nm and 516 nm, respectively, while H2DCF-DA fluorescence was imaged with 483 nm excitation wavelength and 525 nm detection wavelength. Note that each image was taken with a 20 ms exposure time, a 20 objective, and a 10 eyepiece. Ten microliters of each labeled cell suspension was then extracted (diluted in 1 mL PBS) and its mean intensity of fluorescence (MIF) was measured with a fluorescence spectrofluorometer (Varioskan Flash 4.00.51; Thermo Fisher Scientific, Waltham, USA). 2.4. MOM potential monitoring To determine if the mitochondrion is involved in sonoporationinduced stress response, we have examined the MOM potential of sonoporated tobacco cells using the TMRE (tetramethylrhodamine ethyl ester perchlorate) fluorescence dye (ENZ-52309; Enzo Life Sciences, Plymouth, USA). In this analysis, each cell population sample was stained with 80 nM TMRE (dissolved in a 0.2 mM DMSO stock) for 30 min at RT in the dark. After that, the sample was washed with PBS, and the ensuing TMRE fluorescence was observed under a confocal microscope (Fluoview FV1000; Olympus
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Corporation, Tokyo, Japan) using 549 nm and 575 nm, respectively as the excitation and detection wavelengths. Images were taken with a 20 ms exposure time using a 20 objective. The MIF of each TMRE-labeled sample was quantified by extracting 10 lL of the labeled cell suspension (diluted in 1 mL PBS) and placing the extract in a fluorescence spectrofluorometer. 2.5. Cyto-c Western Blot Another mitochondrial stress indicator that we have monitored is the expression level of intra-mitochondrial cyto-c proteins. This was carried out by performing a Western Blot analysis. The procedure first involved isolating the mitochondrial fraction of tobacco cells by removing the cell wall and homogenizing the resultant protoplasts as described in [25]. The mitochondrial fraction was then dissolved in a sodium dodecyl sulfate (SDS) loading buffer [50 mM Tris–hydrogen chloride (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glyercol] at 95 °C for 5 min. After that, the solution was centrifuged at 13,200 rpm for 5 min. to remove insoluble materials. The solubilized mitochondrial supernatants were then separated with 15% SDS–polyarylamide gel electrophoresis, and the results were transferred to a nitrocellulose (NC) membrane. To reveal the amount of loaded proteins, the SDS–polyarylamide gel was stained overnight with a Coomassie brilliant blue solution (0.1% Coomassie brilliant blue R250, 25% isopropanol, 10% glacial acetic acid) and bleached by a destaining solution (5% ethanol, 10% glacial acetic acid). To facilitate blot detection, the NC membrane was blocked with BSA [in TBS (Tris–buffered saline)] at RT for 1 h, and after that it was probed with 1:1250 anti-cyto-c monoclonal antibodies (556433; BD Pharmingen, Franklin Lakes, USA) at 4 °C for overnight. After washing three times with BSA-TBS, the NC membrane was further incubated with secondary goat anti-mouse horseradish peroxidase conjugate antibodies (1:5000) for 1 h at RT (followed by another three rounds of washing afterward). Chemiluminescent detection was subsequently performed to visualize the blots. 2.6. Statistical analysis procedure The sample mean and standard error were computed for all MIF and blot measurements. To determine the statistical significance of the post-sonoporation level of various subcellular stress indicators, the Student t-test was performed to compare the measurements in the sonoporated cell groups against those in the control group. Instances with p values of <0.05 and <0.01 were identified to highlight cases with significant difference. These correspond to 95% and 99% confidence intervals (two-tailed). 3. Results 3.1. Chronic PM depolarization observed in sonoporated cells Fig. 2 shows a set of duplex bright-field/DiBAC4(3) fluorescence images that illustrate changes in the PM potential of tobacco cells immediately after they received sonoporation exposure. In general, it can be seen that the sonoporation exposure has resulted in PM depolarization (Fig. 2b and c). Indeed, the number of cells exhibiting PM depolarization is significantly higher for the case with 0.9 MPa free-field peak rarefaction pressure (Fig. 2c) where more inertial cavitation activities are known to take place (as we demonstrated previously in [5]). This indicates that sonoporation may disrupt the transmembranous ion balance and increase ion transport across the PM. As an analysis of the PM depolarization phenomenon over time, Fig. 3a plots the average DiBAC4(3) fluorescence as a function of
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Fig. 2. PM depolarization of sonoporated tobacco cells. The duplex bright-field/DiBAC4(3) fluorescence image for a cell ensemble with sham exposure is shown in (a). Corresponding results for cells with sonoporation exposure at free-field peak rarefaction pressures of 0.4 MPa and 0.9 MPa are shown in (b) and (c) respectively (immediately after exposure). Stronger green fluorescence indicates less-polarized PM potential (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
0.4 MPa U/S + 1% Microbubbles
0.9 MPa U/S + 1% Microbubbles
a
b
c
d
Normalized to 0.4 MPa Exposure @ 0 h
Fig. 3. Statistical trends of different cellular stress indicators in tobacco cells following sonoporation exposure. Results are shown for free-field peak rarefaction pressures of 0.4 MPa (light gray bars) and 0.9 MPa (dark gray bars) as a function of post-exposure time. The four plots respectively indicate measurements for: (a) PM potential (DiBAC4(3) fluorescence); (b) intracellular ROS (H2DCF-DA fluorescence); (c) MOM potential (TMRE fluorescence); (d) intra-mitochondrial cyto-c expression (Western Blot intensity). Error bars represent standard error of sample mean in all plots (N > 3; and respectively indicate p < 0.05 and p < 0.01).
time for the two exposure groups. According to this figure, PM depolarization remains significant over a multi-hour timeframe (for the 0.9 MPa case, it was over 300% of control level). Such an observation indicates that sonoporation-induced disruption of transmembranous ion balance is not simply a transitory effect. 3.2. Sonoporation may lead to chronic elevation of intracellular ROS Fig. 4 shows H2DCF-DA fluorescence images that depict the intracellular ROS level of sonoporated tobacco cells immediately after exposure. As can be seen, intracellular ROS elevation may be found in sonoporated tobacco cells for the case with acoustic
parameters that favor inertial cavitation activities (see Fig. 4b for the 0.9 MPa case). Note that a transitory elevation of ROS is a central hallmark of a plant cell’s defense response against a stress [16]. As such, our results here suggest that, at higher peak rarefaction pressure levels, the plant cells are more likely to regard sonoporation (after all a physical wounding mechanism) as a significant stress and would activate its defense mechanism to counteract against it. Another point worth noting is that ROS accumulation is known to activate various PM ion channels and thereby mediate the PM potential [17]. This may help explain why PM depolarization is more significant for the 0.9 MPa case (see Fig. 2c).
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Fig. 4. ROS elevation within sonoporated tobacco cells as detected by H2DCF-DA fluoroscopy (stronger green fluorescence indicates higher intracellular ROS level). Results are shown for cells immediately following sonoporation exposure at these free-field peak rarefaction pressures: (a) 0.4 MPa; (b) 0.9 MPa. The fluorescent image for sham exposure is similar to that of (a) and is thus not shown (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Fig. 5. Loss of MOM potential observed in sonoporated tobacco cells. Duplex bright-field/TMRE fluorescence images (illustrating MOM potential level) are shown for cell ensembles 6 h after exposure in the following cases: (a) sham exposure; (b, c) sonoporation exposure with 0.4 MPa and 0.9 MPa free-field peak rarefaction pressure. Loss of MOM potential is marked by weaker red fluorescence in these images.
To analyze the time-lapse impact of sonoporation on the intracellular ROS level, the mean H2DCF-DA intensity was measured from sonoporated cell samples over time (up to 6 h after exposure). Fig. 3b shows the corresponding results for the two exposure groups. It is found that the ROS elevation trend remains persistent over time as similar to that for PM depolarization. This is known to favor activation of PCD by regulating the mitochondrial response [17].
in sonoporated cells. This is supported by quantitative measurements (Fig. 3c) that show a gradual decrease of the TMRE intensity level over time (dropped to less than 70% of control after 6 h). Note that depolarization of the MOM potential is known to lead to mitochondrial membrane permeabilization and it is regarded as a committal step of PCD activation [26]. Our findings thus suggest that the mitochondrion is involved in mediating the stress response in sonoporated plant cells.
3.3. Loss of MOM potential detected in sonoporated cells
3.4. Cyto-c release from mitochondria observed in sonoporated cells
Fig. 5 shows a series of duplex bright-field/TMRE fluorescence images highlighting the MOM potential of sonoporated tobacco cells at 6 h after exposure. The key observation to be noted from these images is the loss of MOM potential in sonoporated cells (Fig. 5b and c), especially for the case with 0.9 MPa free-field peak rarefaction pressure that yields increased inertial cavitation activities. In particular, the red granular TMRE fluorescence as typical of normal mitochondria (Fig. 5a) becomes difficult to trace
One of the key events associated with MOM permeabilization is the release of internal stores of cytotoxic molecules from the mitochondria into the cytoplasm [25]. Our Western Blot analysis has confirmed that this event has indeed occurred (see Fig. 6). For tobacco cells that received 0.9 MPa exposure, they exhibited a significant decrease in the intra-mitochondrial expression of cyto-c proteins that are directly responsible for activating PCD (Fig. 6b). Such a trend is confirmed by the blot intensity statistics shown
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Time After 0.4 MPa U/S Exposure 0h
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Protein Loading
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b
Time After 0.9 MPa U/S Exposure 0h
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types, whilst electrophysiological differences can be seen at the PM where plant cells and mammalian cells have distinct anatomical differences. Since sonoporation is in essence a PM perforation approach, how exactly does this physical stress trigger PM potential changes in plant cells is perhaps one critical question that is worth further investigation in relation to our current work. One possibility we have been hypothesizing is that the physical pores created through this perforation approach would lead to selective activation of plant ion channels and thereby upset the PM ionic balance. It is already known that plant cells tend to respond to a stress by triggering calcium ion influx, and this may in turn trigger the opening of certain PM ion channels including hydrogen, potassium, and chloride [30]. Analysis of these ion channel kinetics would provide indepth insights on the complex interplay of PM electrophysiology in response to sonoporation.
Protein Loading
5. Conclusion Fig. 6. Reduction of intra-mitochondrial stores of cyto-c proteins in sonoporated tobacco cells. (a, b) show the cyto-c Western Blot for the two sonoporation exposure groups (0.4 MPa and 0.9 MPa) at post-exposure time points of 0 h, 3 h, and 6 h. Protein loading was roughly equal in these blots, as indicated by Commassie brilliant blue staining.
in Fig. 3d. Note that cyto-c release into the cytoplasm would block electron transport, and this would further raise the intracellular ROS level to serve as positive feedback for a plant cell’s commitment to PCD [26]. As such, the results here provide further evidence that PCD may be triggered in sonoporated plant cells. 4. Discussion Understanding how sonoporation may adversely affect plant cell homeostasis is of fundamental interest from a biophysical standpoint. In this study, we have sought to address this question by investigating whether a set of subcellular events with recognized roles in stress response mediation are involved in the process. We have generally shown that sonoporation would lead to PM depolarization (Fig. 2) and ROS elevation (Fig. 4). Accompanying with these bioeffects is the initiation of pro-death events from the mitochondrion (Figs. 5 and 6), including MOM permeabilization and release of cyto-c proteins into the cytoplasm (the committal steps of PCD). These findings represent new insights on the biophysical implications of sonoporation, as they illustrate how such a physical stress may disrupt the viability of plant cells. Note that, although BY-2 tobacco cells were used as the plant model of this study, our findings should be readily transferrable to other types of plant cells given that the cytoplasmic pathways regulating plant PCD are by-and-large conserved [26]. From a cell biology perspective, it is interesting to compare the results of this study against those observed for mammalian cells in view of the evolutionary divergence between these two cell types. Some of the findings seem to be in agreement: for instance, ROS elevation and mitochondrial involvement in sonoporation-induced apoptosis have similarly been observed in mammalian cells [27]. In contrast, cell-type variations can be noted for sonoporation’s impact on PM potential; specifically, PM hyperpolarization during sonoporation exposure has instead been reported in mammalian cells [28,29]. These similarities and differences are likely attributed to the fact that the two cell types may have inherited parts of the stress response mechanism from their common unicellular ancestor before developing their own distinct regulation sequence over their separate course of evolution [26]. As such, the mitochondrial response to sonoporation can be expected to be similar since this organelle is known to regulate cellular metabolism in both cell
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Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Subcellular impact of sonoporation on plant cells: Issues to be addressed in ultrasound-mediated gene transfer Peng Qin a, Lin Xu b, Ping Cai c, Yaxin Hu a, Alfred C.H. Yu a,⇑ a
Medical Engineering Program, The University of Hong Kong, Pokfulam, Hong Kong Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China c Department of Instrumentation Science and Engineering, Shanghai Jiaotong University, Shanghai, China b
a r t i c l e
i n f o
Article history: Received 29 May 2012 Received in revised form 31 July 2012 Accepted 1 August 2012 Available online 11 August 2012 Keywords: Sonoporation Tobacco BY-2 cells Plasma membrane Reactive oxygen species Cytochrome-c Programmed cell death
a b s t r a c t Sonoporation (membrane perforation via ultrasonic cavitation) is known to be realizable in plant cells on a reversible basis. However, cell viability may concomitantly be affected over the process, and limited knowledge is now available on how such cytotoxic impact comes about. This work has investigated how sonoporation may affect plant cells at a subcellular level and in turn activate programmed cell death (PCD). Tobacco BY-2 cells were used as the plant model, and sonoporation was applied through a microbubble-mediated approach with 100:1 cell-to-bubble ratio, free-field peak rarefaction pressure of either 0.4 or 0.9 MPa, and 1 MHz ultrasound frequency (administered in pulsed standing-wave mode at 10% duty cycle, 1 kHz pulse repetition frequency, and 1 min duration). Fluoroscopy results showed that sonoporated tobacco cells may undergo plasma membrane depolarization and reactive oxygen species elevation (two cellular disruption events closely connected to PCD). It was also found that the mitochondria of sonoporated tobacco cells may lose their outer membrane potential over time (observed using confocal microscopy) and consequently release stores of cytochrome-c proteins (determined by Western Blotting) into the cytoplasm to activate PCD. These findings provide insight into the underlying mechanisms responsible for sonoporation-induced cytotoxicity in plant cells. They should be taken into account when using this membrane perforation approach for gene transfection applications in plant biotechnology. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction The use of physical means to create transient pores on the plasma membrane (PM) has long been regarded as a non-viral alternative to facilitate gene delivery to biological cells [1,2]. Ultrasound is well-considered as one of the emerging physical approaches, and it generally involves the use of acoustic cavitation principles (i.e. interaction between ultrasound and gaseous cavities) to create microjet forces for PM puncturing on a non-specific and presumably reversible basis [3]. It is now accepted that ultrasound-induced membrane perforation, often referred to as sonoporation [4], may take place even in the presence of a rigid cell wall: a component that is unique to plant cells and not found in mammalian cells [5]. This can potentially benefit the plant biotechnology field, given that gene transfection is an essential step in transgenic crop development [6] and existing transformation techniques based on agrobacterium mediation, particle bombardment, or direct protoplast transformation all have known systemic limitations [7]. Nevertheless, sonoporation as a gene transfection strategy is not yet ready for routine use in plant biotechnology, with one of the main ⇑ Corresponding author. E-mail address: [email protected] (A.C.H. Yu). 1350-4177/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2012.08.008
reasons being that this way of puncturing cells is known to concomitantly affect the ensuing viability of plant cells [5,8]. To account for this issue, it is necessary to gain an in-depth understanding of how sonoporation may bring about lethal impact in plant cells and, thereby, develop strategies to counteract against these deleterious effects. For plant cells, investigations on sonoporation-induced stress response are rather limited at present, and it remains unclear as to why cells may not be able to sustain the pulsed stress brought about by sonoporation. A few early studies conducted using ultrasonic homogenizers and sonicators have provided initial evidence that ultrasound may possibly modify the plant cells’ ion pump activities [9,10] and activate their defense response [11,12]. However, it is inherently difficult to draw a direct correlation between sonoporation and the bioeffects identified in these studies because ultrasonic homogenizers tend to induce a wide range of nonlinear physical phenomena, including shock waves and cavitation [3], on an uncontrollable basis. In this regards, perhaps a more suitable way of investigating the stress response elicited by sonoporation on plant cells would be to make use of a microbubble-mediated sonoporation paradigm where synthetic microbubbles are introduced nearby cell samples to serve as cavitation nuclei and calibrated ultrasound pressure levels are used to trigger acoustic
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cavitation. Such a setup can more systematically facilitate acoustic cavitation whose extent is significantly influenced by the peak rarefaction pressure and microbubble shell properties [13]; indeed, it is already widely adopted for sonoporation studies in mammalian cells [14,15]. Using the microbubble-mediated approach to perform sonoporation, we report here new insights on how sonoporation may affect plant cells at a subcellular level and thereby upset their viability. We have been working with the hypothesis that sonoporation-induced stress response is likely due to the disruption of a chain of cellular physiology events that are known to be involved in maintaining the viability of plant cells. Identification of these cellular disruption events is strategically important because this knowledge can provide essential insights on how we can possibly mediate certain steps in the cytoplasmic signaling cascade to mitigate the attrition issue for ultrasound-based gene transfection in plant cells. Our current work will specifically focus on evaluating the post-sonoporation response of: (1) PM potential, (2) intracellular reactive oxygen species (ROS) level, (3) mitochondrial outer membrane (MOM) potential and (4) cytochrome-c (cyto-c) protein expression. As will be discussed, for these quantities, their deviations from the homeostatic level are known to lead to activation of the programmed cell death (PCD) protocol [16,17]. 2. Materials and methods 2.1. Cell culturing protocol Our investigation was performed on Nicotiana tabacum cells (cultivar Bright Yellow 2) that are well considered as a generic model for higher plant physiology studies [18]. Using the Murashige–Skoog (MS) medium as the culturing medium, the tobacco cells were grown in suspension under a 27 °C dark environment with 120 rpm agitation. The culturing medium was supplemented with sucrose (3%), mono-potassium phosphate (200 mg/L), thiamine (1 mg/L), myo-inositol (100 mg/L), and 2,4-dichlorophenoxy acetic acid (0.2 mg/L; pH 5.8); also, subculturing of cells was performed after every three days. The cells were fostered to grow in the exponential phase before they were harvested for experiments. Note that, prior to receiving sonoporation exposure, the harvested cells were washed three times with phosphate buffered saline (PBS) to remove secondary metabolic products present in the cell samples. 2.2. Sonoporation procedure Sonoporation was applied to the harvested tobacco cells using the exposure apparatus shown in Fig. 1. At the top of this apparatus
is a 15 mm-diameter well plate (176740; Nunc, Rosklide, Denmark) containing a 0.9 mL cell–microbubble mixture (cell density: 106 cells/mL; microbubble volume density: 1% v/v; height of mixture inside well: 5 mm), and ultrasound pulses were transmitted to the well plate from the unfocused transducer that is positioned at the bottom of the water bath. We have previously shown that this exposure setup may bring about sonoporation in BY-2 cells at peak rarefaction ultrasound pressures approaching the microbubble disruption threshold (i.e. when inertial cavitation becomes more significant), but in the process it may concomitantly lead to necrosis and PCD in parts of the cell population [5]. The microbubbles that serve as synthetic cavitation nuclei in this work are essentially lipid-shelled gas bodies. They were fabricated using a perfluorobutane gas sonication procedure as described earlier [19], and their shell material comprises a combination of phosphatidylcholine and polyethylene glycol stearate. Their size ranged from 1 to 5 lm, and the original density in each vial of microbubbles was 106 bubbles/mL. Thus, with a 1% v/v microbubble concentration, the cell-to-bubble ratio was about 100:1 for each experimental trial. Note that we have deliberately chosen to use this microbubble concentration because a high cell-to-bubble ratio (i.e. having more cells than microbubbles) has been previously found to be efficient in facilitating sonoporation without concomitantly inducing significant amounts of cell lysis [20]. In turn, it would allow us to focus more on analyzing the cellular disruption events inside sonoporated cells that remain intact after exposure. The ultrasound transducer used in the exposure setup is of 1-in. diameter and has a 1 MHz center frequency (P1T10W-25; Kunshan Risun Electronic Co., Jiangsu, China). It operated in a tone-burst mode with 1 kHz pulse repetition frequency and 10% duty cycle, and it was driven by a waveform generator (33120A; Agilent Technologies, Palo Alto, USA) with broadband amplification (2100L; Electronics & Innovation Ltd., Rochester, USA). Its excitation voltage was calibrated to generate free-field peak rarefaction pressures of 0.4 or 0.9 MPa at the well-plate region 70 mm away from transducer, as measured using a capsule hydrophone (HGL-0400; Onda Corporation, Sunnyvale, USA). These two pressure levels were chosen as we have previously determined that, for the microbubbles used in our work, 0.4 MPa free-field peak rarefaction pressure predominantly facilitates stable cavitation that generates non-collapsing microbubble pulsations, whilst 0.9 MPa would generate more inertial cavitation activities involving rapid expansion and collapse of microbubbles [5]. Note that, for our exposure apparatus, the actual in situ pressure may be higher since the upper surface of the well plate was coupled to a reflecting air interface that is known to contribute to the presence of standing waves within the cell–microbubble
Tobacco Cells 106 cells/mL density with 104 bubbles/mL Well Plate 70 mm
Signal Amplifier Generator Waveform N-cycle sinusoids, 10% duty cycle 1 kHz pulse repetition freq., 1 min. duration
Water Bath
Transducer Ultrasound 1 MHz frequency 0.4 or 0.9 MPa peak negative pressure
Fig. 1. Illustration of the experimental setup used for sonoporation of tobacco cells. It is based on ultrasound-microbubble mediated cavitation principles, as our team has shown previously [5].
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mixture [21]. Specifically, given that the height of mixture inside the well was 5 mm (equivalent to 3.25 times the acoustic wavelength), approximately 5–6 antinodal planes parallel to the air interface would emerge inside the well plate, and their pressure level may be up to twice the free-field amplitude. In turn, the actual amount of sonoporation activity may be enhanced as a result [22]. It should be emphasized that the acoustic pressure levels used in this study, even for those at the standing-wave antinodal planes, are well-considered as being low such that other nonlinear acoustic phenomena like shock waves and heating would not be inflicted [23]. As well, acoustic cavitation would not be generated at these pressure levels unless synthetic microbubbles are present to serve as artificial cavitation nuclei [24]. Correspondingly, it can be inferred that the bioeffects observed in our study are generally a result of sonoporation generated by ultrasound-microbubble mediated cavitation. We have confirmed this in our preliminary experiments where no subcellular bioeffects were detected in the tobacco cells after ultrasound exposure if synthetic microbubbles were not introduced into the medium (data not shown). 2.3. Monitoring of PM potential and ROS level After the end of exposure, the sonoporated tobacco cells were re-incubated under pre-exposure culturing conditions. To characterize the post-exposure level of PM potential and intracellular ROS, samples in each exposure group were extracted at four different time points (0, 2, 4, and 6 h). These two quantities were respectively labeled in situ using two different fluorescence dyes. PM potential was labeled with the DiBAC4(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] voltage-dependent fluorescence dye (ENZ-52205; Enzo Life Sciences, Plymouth, USA). It was carried out by adding 0.25 lM DiBAC4(3) [buffered in a 2 mM dimethyl sulphoxide (DMSO) stock] to each sonoporated cell population sample (100 lL in volume) that was extracted through PBS washing at 200 g for 5 min. For intracellular ROS, it was tracked with the H2DCF-DA (20 ,70 -dichlorofluorescencein diacetate) fluorescence dye (D-399; Molecular Probes, Eugene, USA). Similar to the DiBAC4(3) labeling procedure, 3 lM of the H2DCF-DA dye was directly added to each sample. Each cell population sample labeled with DiBAC4(3) or H2DCFDA was allowed to further incubate for at least 30 min in a dark, room-temperature (RT) environment with gentle agitation. It was then washed with PBS, and the resulting sample was observed under a differential interference contrast (DIC) microscope (DS-Ri1; Nikon Instruments, Melville, USA). DiBAC4(3) fluorescence was observed using excitation and detection wavelengths of 493 nm and 516 nm, respectively, while H2DCF-DA fluorescence was imaged with 483 nm excitation wavelength and 525 nm detection wavelength. Note that each image was taken with a 20 ms exposure time, a 20 objective, and a 10 eyepiece. Ten microliters of each labeled cell suspension was then extracted (diluted in 1 mL PBS) and its mean intensity of fluorescence (MIF) was measured with a fluorescence spectrofluorometer (Varioskan Flash 4.00.51; Thermo Fisher Scientific, Waltham, USA). 2.4. MOM potential monitoring To determine if the mitochondrion is involved in sonoporationinduced stress response, we have examined the MOM potential of sonoporated tobacco cells using the TMRE (tetramethylrhodamine ethyl ester perchlorate) fluorescence dye (ENZ-52309; Enzo Life Sciences, Plymouth, USA). In this analysis, each cell population sample was stained with 80 nM TMRE (dissolved in a 0.2 mM DMSO stock) for 30 min at RT in the dark. After that, the sample was washed with PBS, and the ensuing TMRE fluorescence was observed under a confocal microscope (Fluoview FV1000; Olympus
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Corporation, Tokyo, Japan) using 549 nm and 575 nm, respectively as the excitation and detection wavelengths. Images were taken with a 20 ms exposure time using a 20 objective. The MIF of each TMRE-labeled sample was quantified by extracting 10 lL of the labeled cell suspension (diluted in 1 mL PBS) and placing the extract in a fluorescence spectrofluorometer. 2.5. Cyto-c Western Blot Another mitochondrial stress indicator that we have monitored is the expression level of intra-mitochondrial cyto-c proteins. This was carried out by performing a Western Blot analysis. The procedure first involved isolating the mitochondrial fraction of tobacco cells by removing the cell wall and homogenizing the resultant protoplasts as described in [25]. The mitochondrial fraction was then dissolved in a sodium dodecyl sulfate (SDS) loading buffer [50 mM Tris–hydrogen chloride (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glyercol] at 95 °C for 5 min. After that, the solution was centrifuged at 13,200 rpm for 5 min. to remove insoluble materials. The solubilized mitochondrial supernatants were then separated with 15% SDS–polyarylamide gel electrophoresis, and the results were transferred to a nitrocellulose (NC) membrane. To reveal the amount of loaded proteins, the SDS–polyarylamide gel was stained overnight with a Coomassie brilliant blue solution (0.1% Coomassie brilliant blue R250, 25% isopropanol, 10% glacial acetic acid) and bleached by a destaining solution (5% ethanol, 10% glacial acetic acid). To facilitate blot detection, the NC membrane was blocked with BSA [in TBS (Tris–buffered saline)] at RT for 1 h, and after that it was probed with 1:1250 anti-cyto-c monoclonal antibodies (556433; BD Pharmingen, Franklin Lakes, USA) at 4 °C for overnight. After washing three times with BSA-TBS, the NC membrane was further incubated with secondary goat anti-mouse horseradish peroxidase conjugate antibodies (1:5000) for 1 h at RT (followed by another three rounds of washing afterward). Chemiluminescent detection was subsequently performed to visualize the blots. 2.6. Statistical analysis procedure The sample mean and standard error were computed for all MIF and blot measurements. To determine the statistical significance of the post-sonoporation level of various subcellular stress indicators, the Student t-test was performed to compare the measurements in the sonoporated cell groups against those in the control group. Instances with p values of <0.05 and <0.01 were identified to highlight cases with significant difference. These correspond to 95% and 99% confidence intervals (two-tailed). 3. Results 3.1. Chronic PM depolarization observed in sonoporated cells Fig. 2 shows a set of duplex bright-field/DiBAC4(3) fluorescence images that illustrate changes in the PM potential of tobacco cells immediately after they received sonoporation exposure. In general, it can be seen that the sonoporation exposure has resulted in PM depolarization (Fig. 2b and c). Indeed, the number of cells exhibiting PM depolarization is significantly higher for the case with 0.9 MPa free-field peak rarefaction pressure (Fig. 2c) where more inertial cavitation activities are known to take place (as we demonstrated previously in [5]). This indicates that sonoporation may disrupt the transmembranous ion balance and increase ion transport across the PM. As an analysis of the PM depolarization phenomenon over time, Fig. 3a plots the average DiBAC4(3) fluorescence as a function of
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Fig. 2. PM depolarization of sonoporated tobacco cells. The duplex bright-field/DiBAC4(3) fluorescence image for a cell ensemble with sham exposure is shown in (a). Corresponding results for cells with sonoporation exposure at free-field peak rarefaction pressures of 0.4 MPa and 0.9 MPa are shown in (b) and (c) respectively (immediately after exposure). Stronger green fluorescence indicates less-polarized PM potential (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
0.4 MPa U/S + 1% Microbubbles
0.9 MPa U/S + 1% Microbubbles
a
b
c
d
Normalized to 0.4 MPa Exposure @ 0 h
Fig. 3. Statistical trends of different cellular stress indicators in tobacco cells following sonoporation exposure. Results are shown for free-field peak rarefaction pressures of 0.4 MPa (light gray bars) and 0.9 MPa (dark gray bars) as a function of post-exposure time. The four plots respectively indicate measurements for: (a) PM potential (DiBAC4(3) fluorescence); (b) intracellular ROS (H2DCF-DA fluorescence); (c) MOM potential (TMRE fluorescence); (d) intra-mitochondrial cyto-c expression (Western Blot intensity). Error bars represent standard error of sample mean in all plots (N > 3; and respectively indicate p < 0.05 and p < 0.01).
time for the two exposure groups. According to this figure, PM depolarization remains significant over a multi-hour timeframe (for the 0.9 MPa case, it was over 300% of control level). Such an observation indicates that sonoporation-induced disruption of transmembranous ion balance is not simply a transitory effect. 3.2. Sonoporation may lead to chronic elevation of intracellular ROS Fig. 4 shows H2DCF-DA fluorescence images that depict the intracellular ROS level of sonoporated tobacco cells immediately after exposure. As can be seen, intracellular ROS elevation may be found in sonoporated tobacco cells for the case with acoustic
parameters that favor inertial cavitation activities (see Fig. 4b for the 0.9 MPa case). Note that a transitory elevation of ROS is a central hallmark of a plant cell’s defense response against a stress [16]. As such, our results here suggest that, at higher peak rarefaction pressure levels, the plant cells are more likely to regard sonoporation (after all a physical wounding mechanism) as a significant stress and would activate its defense mechanism to counteract against it. Another point worth noting is that ROS accumulation is known to activate various PM ion channels and thereby mediate the PM potential [17]. This may help explain why PM depolarization is more significant for the 0.9 MPa case (see Fig. 2c).
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Fig. 4. ROS elevation within sonoporated tobacco cells as detected by H2DCF-DA fluoroscopy (stronger green fluorescence indicates higher intracellular ROS level). Results are shown for cells immediately following sonoporation exposure at these free-field peak rarefaction pressures: (a) 0.4 MPa; (b) 0.9 MPa. The fluorescent image for sham exposure is similar to that of (a) and is thus not shown (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Fig. 5. Loss of MOM potential observed in sonoporated tobacco cells. Duplex bright-field/TMRE fluorescence images (illustrating MOM potential level) are shown for cell ensembles 6 h after exposure in the following cases: (a) sham exposure; (b, c) sonoporation exposure with 0.4 MPa and 0.9 MPa free-field peak rarefaction pressure. Loss of MOM potential is marked by weaker red fluorescence in these images.
To analyze the time-lapse impact of sonoporation on the intracellular ROS level, the mean H2DCF-DA intensity was measured from sonoporated cell samples over time (up to 6 h after exposure). Fig. 3b shows the corresponding results for the two exposure groups. It is found that the ROS elevation trend remains persistent over time as similar to that for PM depolarization. This is known to favor activation of PCD by regulating the mitochondrial response [17].
in sonoporated cells. This is supported by quantitative measurements (Fig. 3c) that show a gradual decrease of the TMRE intensity level over time (dropped to less than 70% of control after 6 h). Note that depolarization of the MOM potential is known to lead to mitochondrial membrane permeabilization and it is regarded as a committal step of PCD activation [26]. Our findings thus suggest that the mitochondrion is involved in mediating the stress response in sonoporated plant cells.
3.3. Loss of MOM potential detected in sonoporated cells
3.4. Cyto-c release from mitochondria observed in sonoporated cells
Fig. 5 shows a series of duplex bright-field/TMRE fluorescence images highlighting the MOM potential of sonoporated tobacco cells at 6 h after exposure. The key observation to be noted from these images is the loss of MOM potential in sonoporated cells (Fig. 5b and c), especially for the case with 0.9 MPa free-field peak rarefaction pressure that yields increased inertial cavitation activities. In particular, the red granular TMRE fluorescence as typical of normal mitochondria (Fig. 5a) becomes difficult to trace
One of the key events associated with MOM permeabilization is the release of internal stores of cytotoxic molecules from the mitochondria into the cytoplasm [25]. Our Western Blot analysis has confirmed that this event has indeed occurred (see Fig. 6). For tobacco cells that received 0.9 MPa exposure, they exhibited a significant decrease in the intra-mitochondrial expression of cyto-c proteins that are directly responsible for activating PCD (Fig. 6b). Such a trend is confirmed by the blot intensity statistics shown
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Time After 0.4 MPa U/S Exposure 0h
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types, whilst electrophysiological differences can be seen at the PM where plant cells and mammalian cells have distinct anatomical differences. Since sonoporation is in essence a PM perforation approach, how exactly does this physical stress trigger PM potential changes in plant cells is perhaps one critical question that is worth further investigation in relation to our current work. One possibility we have been hypothesizing is that the physical pores created through this perforation approach would lead to selective activation of plant ion channels and thereby upset the PM ionic balance. It is already known that plant cells tend to respond to a stress by triggering calcium ion influx, and this may in turn trigger the opening of certain PM ion channels including hydrogen, potassium, and chloride [30]. Analysis of these ion channel kinetics would provide indepth insights on the complex interplay of PM electrophysiology in response to sonoporation.
Protein Loading
5. Conclusion Fig. 6. Reduction of intra-mitochondrial stores of cyto-c proteins in sonoporated tobacco cells. (a, b) show the cyto-c Western Blot for the two sonoporation exposure groups (0.4 MPa and 0.9 MPa) at post-exposure time points of 0 h, 3 h, and 6 h. Protein loading was roughly equal in these blots, as indicated by Commassie brilliant blue staining.
in Fig. 3d. Note that cyto-c release into the cytoplasm would block electron transport, and this would further raise the intracellular ROS level to serve as positive feedback for a plant cell’s commitment to PCD [26]. As such, the results here provide further evidence that PCD may be triggered in sonoporated plant cells. 4. Discussion Understanding how sonoporation may adversely affect plant cell homeostasis is of fundamental interest from a biophysical standpoint. In this study, we have sought to address this question by investigating whether a set of subcellular events with recognized roles in stress response mediation are involved in the process. We have generally shown that sonoporation would lead to PM depolarization (Fig. 2) and ROS elevation (Fig. 4). Accompanying with these bioeffects is the initiation of pro-death events from the mitochondrion (Figs. 5 and 6), including MOM permeabilization and release of cyto-c proteins into the cytoplasm (the committal steps of PCD). These findings represent new insights on the biophysical implications of sonoporation, as they illustrate how such a physical stress may disrupt the viability of plant cells. Note that, although BY-2 tobacco cells were used as the plant model of this study, our findings should be readily transferrable to other types of plant cells given that the cytoplasmic pathways regulating plant PCD are by-and-large conserved [26]. From a cell biology perspective, it is interesting to compare the results of this study against those observed for mammalian cells in view of the evolutionary divergence between these two cell types. Some of the findings seem to be in agreement: for instance, ROS elevation and mitochondrial involvement in sonoporation-induced apoptosis have similarly been observed in mammalian cells [27]. In contrast, cell-type variations can be noted for sonoporation’s impact on PM potential; specifically, PM hyperpolarization during sonoporation exposure has instead been reported in mammalian cells [28,29]. These similarities and differences are likely attributed to the fact that the two cell types may have inherited parts of the stress response mechanism from their common unicellular ancestor before developing their own distinct regulation sequence over their separate course of evolution [26]. As such, the mitochondrial response to sonoporation can be expected to be similar since this organelle is known to regulate cellular metabolism in both cell
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