Preprint submitted to Proc. 6th Intl. Symp.

Therapeutic Ultrasound, Oxford, UK (2006)

Measuring and Modeling Sonoporation Dynamics in Mammalian Cells via Calcium Imaging
R. E. Kumon, P. Parikh, D. Sabens, M. Aehle, D. Kourennyi, C. X. Deng
Department of Biomedical Engineering, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-7207, USA Abstract. In this study, calcium imaging via the fluorescent indicator Fura-2 is used to characterize the sonoporation of Chinese Hamster Ovarian (CHO) cells in the presence of OptisonTM microbubbles. Evolution of the calcium concentration within cells is determined from real-time fluorescence intensity measurements before, during, and after exposure to a 1 MHz ultrasound tone burst (0.2 s, 0.45 MPa). To relate microscopic sonoporation parameters to the measurements, an analytical model that includes sonoporation and plasma membrane transport is developed, assuming rapid mixing (uniform spatial distribution) in the cell. Fitting the measured data to the model provides estimated values for the poration area as a function of poration relaxation rate as well as plasma membrane pump and leakage rates. A modified compartment model that includes the effects of sonoporation, buffering proteins, and transport across the plasma membrane, endoplasmic reticulum, and mitochondria is also investigated. Numerical solutions of this model show a variety of behaviors for the calcium dynamics of the cell. Keywords: Sonoporation, Sonoporation dynamics, Cell membrane, Ultrasound contrast agent, Drug delivery, Gene transfection, Calcium imaging, Whole cell modeling PACS: 43.80.Gx, 43.80.Sh, 87.16.Ac, 87.16.Dg

INTRODUCTION
Sonoporation, the disruption of the cell membrane or pore formation by ultrasound, permits the passage of extracellular agents into the cytoplasm that may not otherwise be permeable through the cell membrane, thereby making it a useful method for drug delivery and gene transfection [e.g., 1-3]. However, real-time monitoring of the movement of such agents has been difficult because of the transient nature and small scale of the sonoporation process. Previous studies in our lab have used voltage clamp techniques to study membrane porosity [4] and duty cycle effects [5] in Xenopus laevis oocytes (frog eggs) and have identified that calcium is necessary for pore resealing after sonoporation. Real-time ratiometric fluorescence imaging has also been used to observe the effects of sonoporation in Chinese Hamster Ovarian (CHO) cells by measuring changes in the intracellular calcium ion concentration [Ca2+] using Fura2AM, a fluorescence indicator [6]. CHO cells are widely used as mammalian expression systems [7] and are used as a representative model system for mammalian cells in this study. Because many interacting factors affect the levels of intracellular

Preprint submitted to Proc. 6th Intl. Symp. Therapeutic Ultrasound, Oxford, UK (2006)

calcium [8], models were constructed to investigate the observed changes in the calcium ion concentration as a result of sonoporation.

EXPERIMENT
As details of the experiment have been described previously [6], only a brief description is provided here. CHO cells were grown in plastic culture dishes following standard protocols until 70% confluency was achieved. Prior to ultrasound exposure, the cells were loaded in darkness with a Fura-2 AM (Invitrogen Molecular Probes F1221, Carlsbad, CA) solution for 60 min. Fura-2 AM is a fluorophore that rapidly binds to Ca2+ in a 1:1 ratio and shifts its peak absorbance from 380 to 340 nm upon binding. The cells were then washed and immersed in a phosphate-buffered solution (PBS) with [Ca2+] (0.9 to 2.5 mM) much greater than the intracellular [Ca2+] (<1 M). The cells were then treated with a 0.2 s tone burst with a center frequency of 1 MHz and amplitude of 0.45 MPa in the presence of a 5% solution of the ultrasound contrast agent OptisonTM to facilitate sonoporation. For ratiometric fluorescent imaging, the cells were excited alternatingly with light at 380 nm and 340 nm (rapid switching between the two wavelengths of excitation), and photomicrographic images were taken with an inverted microscope and Nikon Cool CCD camera at 510 nm after each exposure. Images were acquired at every 1 to 2 s for 10 min. The fluorescence intensity at both excitation wavelengths for each cell was determined by averaging the measured intensities over the area of the cell using the image analysis routines of MetaFluorTM (Molecular Devices, Downingtown, PA). Conversion of fluorescence intensities to concentration values is performed according to the relation [9] R Rmin , (1) [Ca 2+ ] = K d Q Rmax R
bg bg where R = ( F340 F340 )/( F380 F380 ) is the ratio of the background-corrected fluorescence intensity F340 after excitation at 340 nm with corresponding intensity

F380 after excitation at 380 nm, the parameters Rmax = 3.33 , Q = 4.94 were obtained by using a Fura-2 calibration kit (Invitrogen Molecular Probes F6774) in a solution without cells, K d = 135 nM is the dissociation constant for Fura-2 with Ca2+ [9], and Rmin = 0.01 is taken to be a small but nonzero value. The ratio of intensities is used because it is less sensitive to artifacts due to indicator concentration, photobleaching, and path length [9]. The aforementioned extracellular calibration parameters were then used as estimates for the intracellular calibration parameters, which may differ by viscosity and pH induced corrections [10]. Figure 1 shows results for a typical cell before and after the application of ultrasound (left vertical dashed line) where the extracellular [Ca2+]=2.5 mM. Figure 1(a) shows the background fluorescence intensities in a region adjacent to the cell as a function of time. The average intensities at 380 nm and 340 nm are shown by the horizontal dashed lines and are also indicated in Fig. 1(b), which shows the corresponding intensities inside the cell. When sonoporation occurs, the high extracellular [Ca2+] results in the transport of calcium into the cell. As a result, more of
2

Preprint submitted to Proc. 6th Intl. Symp. Therapeutic Ultrasound, Oxford, UK (2006)

the pre-loaded Fura-2 binds with Ca2+, causing the intensity due to excitation at 380 nm to decrease (less unbound Fura-2) and the intensity due to excitation at 340 nm to increase (more bound Fura-2). Figure 1(c) shows the change in fluorescence intensities after background correction, while Fig. 1(d) shows the corresponding ratio of these intensities (340 nm to 380 nm).
220 218 216 Fluorescence Intensity 214 212 210 208 206

(a)
F380 F340 Fluorescence Intensity

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F380 F380 F340 F340
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FIGURE 1. (a) Background fluorescence emission intensities at 510 nm due to excitation at 380 nm (top line) and 340 nm (bottom line) as a function of time. (b) Intracellular fluorescence emission intensities at 510 nm due to excitation at 380 nm (top line) and 340 nm (bottom line) as a function of time. Application of ultrasound occurred at the time indicated by the large arrows. The right vertical dashed line represents the approximate time of return to equilibrium. (c) Intracellular fluorescence intensities after background correction. (d) Ratio of fluorescence intensities (340 nm to 380 nm) after background correction.

ANALYTICAL MODEL
To understand the relationship between the poration parameters and the macroscopic evolution of the intracellular [Ca2+], a basic compartment model is constructed to describe the [Ca2+] inside the cell with linearized leakage and transport terms[10]. If sonoporation occurs instantly and then relaxes with exponential decay, it follows that the time evolution of the cytosolic [Ca2+] is described by dc PM PM = J leak J pump + J sp = k1 (ce c) k2 c + (ace c) S0 e 0t , (2) dt

Preprint submitted to Proc. 6th Intl. Symp. Therapeutic Ultrasound, Oxford, UK (2006)

where ce is the extracellular [Ca2+], k1 and k2 are the rate constants for leakage and pumping through the plasma membrane, = D / Vcell h is the pore transport rate per unit area, D is the ion diffusion coefficient, Vcell is the cell volume, h is the membrane thickness, S0 is the initial poration area, and 0 is the poration relaxation rate, a = 1 + zeVm / k BT is a electrochemical diffusion factor derived from the Nernst-Planck equation, z is the valency of the ion (signed value), e is the magnitude of the electric charge, Vm = e is the membrane potential difference, kB is the Boltzmann constant, and T is temperature. Representing the whole cell by a single concentration value is reasonable provided that there is minimal spatial variation or spatial inhomogeneities dissipate much faster than the post-sonoporation relaxation. Eq. (2) has the exact solution k c (t ) S ac (t ) k c S ac c(t ) = 1 e 1 + 0 e 2 e 0t + ci 0 1 e 1 0 e 2 e1t e (exp[ 0t ]1) , (3) 1 2 1 2 where 1 = k1 + k2 , 2 = 1 0 , = S0 / 0 , m (t ) = 1 F1 (1,1 m / 0 , e 0t ) is the Kummer confluent hypergeometric function, m = m (t = 0) for m = 1, 2 , and
B

ci 0 = c(0) . In the limit of << 1 , Eq. (2) has an approximate solution of the form capp (t ) = c +

S0 ace t S0 ace ( k + k e + ci 0 c e k1 + k2 0 k1 + k2 0
0 1

2 )t

(4)

where c = k1ce /(k1 + k2 ) is the equilibrium concentration as t . Eq. (4) has a biexponential form, with the decay of the exponentials determined by the poration relaxation rate and total leakage and pump rate. The data in Fig. 1 shows that c = ci 0 , and hence the data was fit to the form of Eq. (4) subject to this requirement, using D = 790 m 2 /s [11], Vcell = 4200 m 3 (if spherical, rcell = 10 m ), h = 10 nm , z = +2 , Vm = 10 mV [6], T = 293 K , and ce = 2.5 mM . Although no direct measurements of pore relaxation in sonoporation in mammalian cells are available currently, it has been suggested that the poration relaxation rate is probably greater than 1 s1 [2, 4], the time resolution of this imaging data was insufficient to reliably use 0 as a fitting parameter. Instead, a series of least-squares fits were performed using the three fitting parameters k2 , ci 0 , S0 with various assumed values of poration relaxation rate in the range 0.1 s 1 0 10 s 1 . The leastsquares fit for 0 = 10 s 1 , shown in Fig. 2(a), yielded k 2 = 0.026 s 1 , ci 0 =16 nM, and
S 0 = 870 nm 2 . With the assumption c = ci 0 , it also follows that k1 = k2 /(ce / ci 0 1) =

1.6 107 s 1 . Similar values resulted for all 0 1 s 1 . The relationship between the poration relaxation rate 0 and poration area S0 is nearly linear, as shown in Fig. 2(b). In all cases, the use of the approximate solution of Eq. (4) is justified because 0.002 << 1 . Physically, this means that the ratio of the diffusion transport rate S0 to the poration relaxation rate 0 is small. The leakage and pump

Preprint submitted to Proc. 6th Intl. Symp. Therapeutic Ultrasound, Oxford, UK (2006)

rates are comparable to those for bullfrog sympathetic neurons ( k1 = 3.7 10 7 s 1 ,

k 2 = 0.0335 s 1 for c < 600 nM [8]; k1 = 5 106 s 1 , k 2 = 0.132 s 1 [11]). The initial

concentration ci 0 is probably low, but this value is probably a result of the uncertainty in Rmin for this data. If the area is assumed to be from a single circular pore, the effective range of radii corresponding to Fig. 2(b) is 1.6 nm r0 16 nm, which is in the vicinity of reported estimates based on the uptake of markers.
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FIGURE 2. (a) Ratio of background-corrected fluorescence intensities from above (black) and best fit to model (red) for poration relaxation rate 0=10 s1. (b) Relationship between best-fit poration area for various specified poration relaxation rates.

NUMERICAL MODEL
In real cells, calcium ions can be stored in organelles and buffered by proteins. To model these effects, flux terms were added to Eq. (2) for transport across the endoplasmic reticulum (ER) and mitochondra (M) and for binding to a protein (Pr) in the cytosol (Cyt), thus yielding a four compartment model, as illustrated schematically in Fig. 3. The nonlinear model equation for the cytosolic [Ca2+] is dcCyt PM PM ER ER ER M M Pr Pr = J leak J pump + J sp + J ch + J leak J pump + J out J in + J out J in , (5) dt where the added terms are taken from [12], along with three additional differential equations for the evolution of [Ca2+] in the ER and mitochondria, and Ca2+protein binding kinetics. The resulting system of ODEs was solved using the Runge-Kutta integration routines in MATLAB (Mathworks, Natick, MA). The ensuing model has 16 parameters and 3 initial conditions in addition to the parameters already described in the analytical model, all of which were also taken from [12]. Although these parameters are not specific to CHO cells, this initial simulation study is useful to show examples of the types of behaviors that can be exhibited in cells via various mechanisms, including the complex calcium oscillations demonstrated in the original model [12]. Figure 4 shows the results of three such simulations superimposed on the measured data in Fig. 3(a), using the same calibration as above to convert the calcium ion

Preprint submitted to Proc. 6th Intl. Symp. Therapeutic Ultrasound, Oxford, UK (2006)

CICR

CICR

J sp

M out

M in

ccyt = [Ca 2 + ]cyt

J
Pr

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Pr

ER J leak

[Ca ]ext

2+

CaPr

Pr+Ca

2+

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PM J pump

FIGURE 3. Schematic diagram of four compartment model. Flux terms derive from sonoporation (sp), mitochondria (M), protein buffering (Pr), endoplasmic reticulum (ER), and plasma membrane (PM). Several terms exhibit nonlinear calcium-induced calcium released with respect to the cytosolic calcium concentration.

concentration to ratiometric form. In each case, k1 = 1.6 10 7 s 1 , k 2 = 0.026 s 1 ,

ci 0 =16 nM, 0 = 10 s 1 , S 0 = 870 nm 2 , to allow comparison to the fitted curve in Fig. 3(a). First, consider the effect of including PM and ER transport only, as shown by the dashed line in Fig. 4(a). The ratio rises rapidly upon sonoporation followed by slower decay than in Fig. 3(a) with PM transport only. The initial influx of calcium ions is slowly pumped out of cell and into the ER until the ER channel closes and then nearly all remaining calcium is rapidly sequestered. This latter result is a nonlinear effect of cytosolic calcium-induced changes in the rate constant of the channel. Next, consider the effect of adding in the mitochondria, as shown by the dotted line in Fig. 4(a). Again the ratio rises rapidly due to sonoporation, but it is then followed by rapid drop due to pumping into ER. As compared with the PM+ER case, an even slower decay occurs as calcium ions are slowly pumped out of cell and into both the ER and mitochondria. The nonlinear ER channel eventually closes and then nearly all remaining calcium is rapidly sequestered. Finally, consider the effect of adding in the buffering proteins, as shown in Fig. 4(b). The ratio rises rapidly initially but is then
3.5 Experiment Theory (PM+ER) Theory (PM+ER+M)
3.5 Experiment Theory (PM+ER+M+Pr)

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FIGURE 4. Comparison of ratiometric measurements with simulations (non-CHO cell specific parameters) for (a) PM+ER model [dashed] and PM+ER+M model [dotted] and (b) PM+ER+M+Pr model.

Preprint submitted to Proc. 6th Intl. Symp. Therapeutic Ultrasound, Oxford, UK (2006)

followed by complicated set of small oscillations (similar to [8]) interrupted by larger oscillations, as a complicated exchange of calcium occurs between the various compartments in the cell. Additional calculations indicate a slow net sequestration of calcium into the mitochondria and net release of calcium from the buffering proteins during the course of the oscillations. This mechanism may be responsible for the significant oscillations seen subsequent to the initial peak in the measured data.

CONCLUSIONS
Ratiometric fluorescence measurements of cells exposed to high levels of extracellular calcium show a rapid rise in [Ca2+] followed by a longer decay back to the original level. An analytical whole cell model was derived to relate the measured data to sonoporation and cell parameters. Fitting to this model yields reasonable values for the plasma membrane pump and leakage parameters as well as poration areas for specified poration relaxation rates. Numerical solutions of a model with additional nonlinear terms to represent the ER, mitochondria, and protein binding show that a variety of complicated behavior are possible, but additional work is needed to determine the specific cellular parameters appropriate for CHO cells to fit the model to the observed results.

ACKNOWLEDGMENTS
This work was supported by the Presidential Research Initiative at Case Western Reserve University and funding from the National Cancer Institute of the U.S. National Institutes of Health. The authors would also like to thank Yun Zhou for technical assistance and Olivier Izad for cell culturing.

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