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Different effects of sonoporation on cell morphology and viability.

INTRODUCTION

Conventional drug delivery systems, such as systemic administration via intravenous injection or oral administration, are often not sufficient for delivery of therapeutic compounds such as proteins and genes [1, 2]. A recent development in delivery systems for therapeutic compounds is ultrasound (US)-aided intracellular delivery [3-5]. It has been demonstrated that US can achieve efficient intracellular delivery of a variety of drugs and/or genes [6-8]. Sonoporation is defined as the formation of transient, nonspecific pores or openings in the cellular membranes upon US exposure was commonly considered as the main mechanism of action for efficient drug delivery [9-11]. However, several studies have recently reported heterogeneity in the levels of both small- and macro- molecular uptake by sonoporation [12-14]. Cells with various levels of molecular uptake can be generally divided into two groups: cells with high levels of molecular uptake and those with low levels of molecular uptake. The exact mechanism is still not fully understood. Zarnitsyn et al. [15] presented a theoretical model that determined membrane pore size as a function of calcein (a cell impermeant dye) uptake where calcein uptake is directly related to pore size (i.e. greatest calcein uptake in cells with the largest pores). In the current study, US was applied to adherent cells in the cell culture dishes in order to establish a model of heterogeneity in sonoporation. The possible mechanism of action was studied by observing changes in cell morphology immediately after sonoporation using scanning electron microscope (SEM) and cell viability immediately and 6 h after sonoporation using fluorescence microscope.

MATERIALS AND METHODS

Cell lines

Human prostate cancer DU145 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured as monolayers and grown to 80% confluence on cell culture dishes (35 mm in diameter) in RPMI-1640 media (GIBCO, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; GIBCO, USA), 2 mmol/L glutamine, 100 IU/ mL penicillin, 100 [micro]g/mL streptomycin, and 10 mmol/L HEPS (pH 7.4) at 370C, 5% C[O.sub.2], and 90% relative humidity.

[FIGURE 1 OMITTED]

Cell pre-treatment

Three ml cell culture media (fresh RPMI-1640 with 10% FBS) containing 5% (v/v) of the microbubble contrast agent-Sonovue (Bracco International B.V., Italy) and 10 [micro]M calcein (623 Da, radius=0.6 nm; A green fluorescent and cell membrane impermeant stain, Sigma, USA) was added into the cell culture dishes containing adherent human prostate cancer DU145 cells before sonication.

Ultrasound apparatus and exposure

Ultrasound was generated at 21 kHz by a function generator and amplifier (Shanghai Institute of Ultrasound in Medicine, Shanghai, China) that controlled the transducer via matching transformer (Shanghai Institute of Ultrasound in Medicine, Shanghai, China). The transducer was calibrated using laser interferometry as described by Wu et al. [16]. Acoustic power of 10 mW, 100% duty cycle and 1 s exposure time were chosen for sonication treatment. Transducer tip (flat and round with a diameter of 13 mm) was fixed by a holder and faced vertically upwards. A cell culture dish was placed just above the transducer surface with a thin layer of gel between them (Figure 1).

Cell morphology observation

To view cell morphology, we imaged adherent cells using scanning electron microscope (SEM) (Quanta 200, Philips, Netherlands). Briefly, before sonication 3 ml of fresh cell media (RPMI-1640 with 10% FBS) containing 5% (v/v) of the microbubble contrast agent-Sonovue and 10 [micro]M calcein, was added into the cell culture dish containing adherent human prostate cancer DU145 cells. Immediately (5 sec after sonication) cell culture media was discarded and 3 ml of 2% EMgrade glutaraldehyde (Sigma, USA) was added. Preparations for SEM were performed using established techniques.

Cellular viability assessment

To identify cellular viability, propidium iodide (PI) (Sigma, USA), which is able to stain the nuclei of nonviable, membrane-compromised cells with red fluorescence, was added to the cell culture dishes containing adherent human prostate cancer DU145 cells 5 min after sonication producing a final concentration of 1 [micro]M. Propidium iodide (PI) was left on cells for a total of 10 min at room temperature, thereafter the cell culture media containing PI and calcein was removed and cells were washed twice with phosphate buffer solution (PBS; GIBCO, USA) and 1 ml of fresh RPMI-1640 cell culture media (with 10% FBS) was added prior to being assayed by fluorescence microscope (ZX70, OLYMPUS, Japan). Merged image was used to show PI staining (red) of cell with calcein uptake (green).

RESULTS

Levels of intracellular calcein uptake immediately after sonoporation

Several studies have reported that US exposure on adherent cells in the presence of microbubble contrast agents can induce cell detachment and sonoporation [17, 18]. Consistent with these studies, the substrate was partially cleared of cells following US exposure in the presence of the microbubble contrast agent-Sonovue (Figure 2A). Adherent cells, which have not been washed away but line the border between occupied and empty region, emitted green fluorescence under fluorescence microscope duo to the uptake of calcein (Figure 2B). No green fluorescence was detected for cells far way from the sonciation-induced detachment (Figure 2B). It was also shown that cells with calcein uptake can be roughly divided into two subgroups: cells with high levels of calcein uptake (strong green fluorescence, Figure 2B) and cells with low levels of calcein uptake (weak green fluorescence, Figure 2B). Changes in cell morphology immediately after sonoporation By using scanning electron microscope (SEM), it was shown that cells far away from the vacanted regions displayed rich and homogeneous distribution of microvilli on the cellular surfaces (Figure 3A). While, various changes in cell surface morphology for those cells surrounding the vacanted regions could be detected: smooth surface (Figure 3B), pores in membrane (Figure 3C) and irregular cell surface (Figure 3D).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Changes in cell viability after sonoporation

Under fluorescence microscope, it was shown that immediately after sonoporation, both groups of cells with high levels of calcein uptake and low levels of calcein uptake were viable as evidenced by no staining with PI (Figure 4A); 6 h after sonoporation, group of cells with low levels of calcein uptake still remained viable (Figure 4A-B, negative PI staining as indicated by the long arrow), while group of cells with high levels of calcein uptake died (Figure 4A-B, PI staining changed from negative into positive as indicated by the short arrow).

DISCUSSION

This study showed different intracellular calcein uptake and changes in cell morphology and viability after sonoporation. Furthermore, group of cells with low levels of calcein uptake remained viable 6 h after sonoporation, while group of cells with high levels of calcein uptake died. Sonoporation is defined as formation of transient, nonspecific pores or openings in the cellular membranes upon US exposure [19]. Acoustic cavitation produced by US exposure is believed to be the main physical mechanism caused by US exposure [20, 21]. Acoustic cavitation is the process entailing bubbles formation, growth, vibration or even collapse in the medium under US activation [22]. Microbubble contrast agents could act as the acoustic cavitation nuclei and produce acoustic cavitation under ultrasound exposure [23]. It is currently believed that mechanical wounding on cells due to acoustic cavitation is the predominant mechanism of action of sonoporation [10]. Several studies have investigated the changes in cell morphology immediately after sonication [10, 19, 24]. However, results vary and most are focused on observing the pores in the membrane. In our study, we observed different changes in cell morphology immediately after sonoporation; these included: smooth surface, pores in the membrane and irregular cell surface. It is noteworthy that in our study smooth surface was the most common change while pores were rarely seen. One possible explanation is that the pores may have been resealed before cell fixation [25]. Our study further showed that some cells containing high levels of calcein uptake died 6 h after sonoporation; while those with low levels of calcein uptake survived. It has been reported that fractions of cells with various levels of molecular uptake of calcein can be affected by changing the US exposure intensity [12]. Therefore, combined with the study by Zarnitsyn et al. [15], it is suggested that levels of molecular uptake of calcein may be consistent with the levels of cell membrane wounds . The exact mechanism regarding how cellular membrane wounding results in delayed cellular death is still unknown. Several studies have reported that there is an immediate calcium ion influx following mechanical wounding (including sonication) [26, 27]. Hutcheson et al. [13] successfully rescued up to 44% of cells with high levels of calcein uptake from apoptosis by the addition of a calcium ionic chelator. However, several studies also show that immediate calcium ion influx after sonication is vital to the wound repairing [26-28]. Therefore, calcium ion influx after sonication may play a complex role in sonoporation [29]. However, there is still one main limitation in our study since we did not measure the accurate distribution of the acoustic field in our experimental setup. Nonetheless, the aim of our study is not to optimize the exposure parameters, but to observe the changes in cell morphology of cells with molecular uptake under this simple experimental model [30, 31].

[FIGURE 4 OMITTED]

CONCLUSION

Our study showed different effects of sonoporation on intracellular calcein uptake, cell morphology and viability. It was suggested that various changes in cell morphology may be responsible for different levels of intracellular calcein uptake and changes in cell viability after sonoporation.

Submitted: 15 September 2011 / Accepted: 19 April 2012

ACKNOWLEDGEMENTS

The authors would like to thank the following Professors: Qian Cheng (Institute of Acoustics, Tongji University, Shanghai, China) for ultrasound parameters calibration and Wen-de Shou (Shanghai Institute of Ultrasound in Medicine, Shanghai, China) for acoustic theory consultation.

DECLARATION OF INTEREST

This work was supported in part by the National Natural Science Foundation of China (grant # 30770562) and Shanghai Science and the Technology Committee Basic Research Program (grant #10JC1412600). All authors have read and approved this manuscript. Neither the submitted paper nor any similar paper, in whole or in part has been or will be published in any other primary scientific journal. No conflict of interest exists in the submission of this manuscript.

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Ji-Zhen Zhang (1), Jasdeep K. Saggar (2), Zhao-Li Zhou (3), Bing-Hu (1) *

(1) Department of Ultrasound In Medicine, Shanghai Jiao Tong University Affiliated 6th People's Hospital, Shanghai Institute of Ultrasound in Medicine, 600 Yi Shan Road, Shanghai 200233, China. (2) Department of Medical Biophysics, University of Toronto, 610 University Avenue (Room 10-610), Toronto, Ontario, Canada M5S 3E2. (3) Central Research Institute, Shanghai, Pharmaceuticals Holding Co., Ltd. Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

* Corresponding author: Bing-Hu, Department of Ultrasound In Medicine, Shanghai Jiao Tong University Affiliated 6th People's Hospital, Shanghai Institute of Ultrasound in Medicine, 600 Yi Shan Road, Shanghai 200233, China. Tel: 086-21-64369181; Fax: 086-21-54488254 e-mail: binghuzz@sina.cn
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Author:Zhang, Ji-Zhen; Saggar, Jasdeep K.; Zhou, Zhao-Li; Bing-Hu
Publication:Bosnian Journal of Basic Medical Sciences
Article Type:Report
Geographic Code:4EXBO
Date:May 1, 2012
Words:2628
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