ultrasound-mediated disruption of cell membranes. ii....

Ultrasound-mediated disruption of cell membranes. II.Heterogeneous effects on cells

Hector R. Guzman, Daniel X. Nguyen, Sohail Khan, and Mark R. Prausnitza)

School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0100

~Received 18 August 2000; accepted for publication 4 April 2001!

Ultrasound has been shown to reversibly and irreversibly disrupt membranes of viable cells througha mechanism believed to involve cavitation. Because cavitation is both temporally and spatiallyheterogeneous, flow cytometry was used to identify and quantify heterogeneity in the effects ofultrasound on molecular uptake and cell viability on a cell-by-cell basis for suspensions of DU145prostate cancer and aortic smooth muscle cells exposed to varying peak negative acoustic pressures~0.6–3.0 MPa!, exposure times~120–2000 ms!, and pulse lengths~0.02–60 ms! in the presence ofOptison~1.7% v/v! contrast agent. Cell-to-cell heterogeneity was observed at all conditions studiedand was classified into three subpopulations: nominal uptake~NUP!, low uptake~LUP!, and highuptake ~HUP! populations. The average number of molecules within each subpopulation wasgenerally constant: 104– 105 molecules/cell in NUP,;106 molecules/cell in LUP, and;107

molecules/cell in HUP. However, the fraction of cells within each subpopulation showed a strongdependence on both acoustic pressure and exposure time. Varying pulse length produced nosignificant effect. The distribution of cells among the three subpopulations correlated with acousticenergy exposure, which suggests that energy exposure may govern the ability of ultrasound toinduce bioeffects by a nonthermal mechanism. ©2001 Acoustical Society of America.@DOI: 10.1121/1.1376130#

PACS numbers: 43.80.Gx, 43.80.Sh@FD#

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I. INTRODUCTION

Drug delivery and gene therapy are limited by the neto deliver large numbers of molecules into living cells. Aspossible solution, ultrasound has been shown to enhancelecular transport across cell membranes through a menism believed to involve acoustic cavitation. The abilityreversibly increase cell membrane permeability has beenserved in studies using small molecules, macromolecuand genetic material~Fechheimeret al., 1986; Saad andHahn, 1987; Harrisonet al., 1996; Baoet al., 1997; Green-leaf et al., 1998; Miller et al., 1999; Guzma´n et al., 2001!.However, these and other studies have generally not qufied molecular uptake within affected cells on a cell-by-cbasis. This type of analysis would provide absolute numbof molecules within cells, which is important for drug angene delivery applications and to support quantitative meling efforts. It would also identify and quantify heterogenity in ultrasound’s effects, which is important for applictions where uniform responses among a population of cmay be desirable.

In this study, molecular uptake and cell viability aquantified on a cell-by-cell basis for large numbers of invidual cells~e.g., 20 000 cells per sample! using flow cytom-etry. We hypothesize that within a population of cells socated under the same conditions, the number of molecdelivered into each cell will be highly variable. To test thhypothesis, data from a companion study~Guzman et al.,2001! are reanalyzed to quantify levels of molecular uptaon a cell-by-cell basis. This re-analysis permits identificat

a!Electronic mail: [email protected]

J. Acoust. Soc. Am. 110 (1), July 2001 0001-4966/2001/110(1)/5

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of distributions in uptake and provides a means to quanany observed heterogeneity.

Our companion study~Guzman et al., 2001!, whichmeasured the effects of acoustic pressure, exposure timepulse length on cell viability and average molecular uptawithin cells, concluded that these bioeffects correlated wacoustic energy exposure over the conditions tested.therefore propose as a second hypothesis for the prestudy that cell-to-cell heterogeneity in molecular uptakesulting from sonication will correlate with acoustic energexposure.

It is not clear a priori that cell-to-cell heterogeneityshould be expected, which in part motivates this study.example, molecular uptake induced by an electrical metto increase cell membrane permeability—electroporationhas been shown to be homogeneous over a broad rangexperimental conditions in mammalian cells~Prausnitzet al.,1993; Canatellaet al., 2001! and can show bimodal distributions in yeast~Gift and Weaver, 1995!. The observed homogeneity can be attributed to the uniform electric field expeenced by cells during electroporation, whereasheterogeneity may be due to the nonspherical shape of ycells that results in different transmembrane voltages afunction of cell orientation. In contrast, ultrasound’s bioefects are generally attributed to cavitation, which is hetegeneous in both time and space~Leighton, 1994; Milleret al., 1996; Barnett, 1998!. For this reason, we have hypothesized that each cell will experience different interactiowith cavitation bubbles, which thereby result in different leels of bioeffects on a cell-to-cell basis.

59797/10/$18.00 © 2001 Acoustical Society of America

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II. MATERIALS AND METHODS

To measure possible heterogeneity in the effects oftrasound on cells, we used data collected in a companstudy ~Guzman et al., 2001! which quantified molecular uptake and cell viability over a range of peak negative acoupressures~0.6–3.0 MPa!, exposure times~120–2000 ms!,and pulse lengths~0.02–60 ms!. Detailed descriptions of celculture, experimental procedures, ultrasound application,data analysis are presented in that study. The followingsummary of those experimental methods, as well as atailed description of additional analysis used in this study

A. Experimental methods

DU145 prostate cancer cells~DU145! or human aorticsmooth muscle cells~AoSMC! grown as monolayers werharvested and resuspended to a concentration of 13106

cells/ml in Dulbecco’s phosphate buffered saline. Calc~623 Da, radius50.6 nm!, a molecule simulating a smadrug, was added at a concentration of 10mM and Optisoncontrast agent, which provides nuclei for cavitation, wadded to achieve a final concentration of 1.7% v/v. Csamples were then exposed to ultrasound at the conditdescribed previously using focused 500-kHz ultrasoundthe apparatus described previously~Guzman et al., 2001!.Using flow cytometry, molecular uptake caused by ultsound was determined by measuring the intensity of grfluorescence emitted by calcein taken up by cells. Cellability was determined by measuring the intensity of rfluorescence emitted by propidium iodide added as a viaity stain to cell samples after sonication~Guzman et al.,2001!.

B. Flow cytometry analysis of heterogeneity

Intracellular calcein fluorescence was measured usflow cytometry; 20 000 cell measurements were collectedsample to ensure that a statistically significant cell popution was analyzed. As shown in Fig. 1, the fluorescencetensity of each sample of cells can be presented as a hgram. In a representative control sample of cells unexpoto ultrasound@Fig. 1~a!#, a single population of cells ispresent with a distribution of fluorescence most likely dueautofluorescence, optical and electrical noise, and low-lesurface binding of calcein. In representative samples of cexposed to ultrasound@Figs. 1~b!–~d!#, the histograms showbroad, heterogeneous distributions of cell fluorescenwhich appear to contain multiple subpopulations. The shobserved in these histograms, i.e., two peaks and a wvalley in between, was common to almost all cell sampexposed to ultrasound. For this reason, the observed hegeneity was analyzed using three subpopulations of cellsshown pictorially in Fig. 1~c!.

To analyze the distributions of viable cells containwithin each sample, raw data from histograms generateWINMDI ~TSRI Flow Cytometry, San Diego, CA!, were ex-ported intoEXCEL ~Microsoft, Redmond, WA! as ASCII filesusing the utility LDATA ~Robinson and Kelly, 1998!. InEXCEL, the data were formatted and then exported intoMIX

Software 3.1 ~Ichthus Data Systems, Hamilton, Ontari

598 J. Acoust. Soc. Am., Vol. 110, No. 1, July 2001

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Canada!. MIX is a statistical software package used to alyze populations containing a mixture of subpopulations. Uing MIX , we found the best fit for a set of three normdistributions to describe the uptake histogram for easample exposed to ultrasound@Fig. 1~c!#. MIX then calculatedthe fraction of cells, the fluorescence mean, and the standeviation of the fluorescence mean for each of the threesubpopulations. Molecular uptake in each subpopulationmeasured by subtracting the mean fluorescence of [email protected]., Fig. 1~a!# from the mean fluorescence of eacsubpopulation in each of the exposed samples. The numof calcein molecules delivered per cell was then determifrom these mean fluorescence measurements, as descpreviously~Guzman et al., 2001!.

C. Visual verification of heterogeneity

To visualize molecular uptake into cells, a ZeiLSM510 confocal microscope~Carl Zeiss, Thornwood, NY!was used to image the fluorescence emitted from cellsposed to 488-nm argon UV lasers~optical section at;8-mmpenetration depth, which is near the center of each c!.Green fluorescence~calcein! indicated molecular uptake, refluorescence~propidium iodide! stained the nuclei of deadcells, and blue fluorescence~Hoechst 33342; MoleculaProbes, catalog no. H-1399! nonspecifically identified the

FIG. 1. Fluorescence histograms of cell samples showing uptake of ca~20 000 cells/sample!. ~A! Fluorescence of a control sample showGaussian-distributed background fluorescence in the first decade of thetogram.~C! Fluorescence signal in the exposed sample is heterogeneoucan be divided into regions termed nominal~NUP!, low ~LUP!, and high~HUP! uptake populations~60-ms pulse length, 540-ms exposure time, a3.0 MPa peak negative pressure!. ~B, D! Histograms showing that at otheultrasound conditions there still exist three subpopulations, but with difent relative numbers of cells in each sub-population~60-ms pulse length, 2-sexposure time, and 1.2~B! and 2.4~D! MPa peak negative pressure!.

Guzman et al.: Heterogeneous bioeffects caused by ultrasound

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nuclei of all cells. Confocal images were used to visuacorroborate the heterogeneity observed in flow cytomedata.

D. Statistical analysis

At each condition tested, a minimum of three replicacell samples was measured, from which the mean and sdard error were calculated. A Student’s t-test was used wcomparing two experimental conditions and a one-wanalysis of variance~ANOVA a50.05! was performedwhen comparing one factor with three or more experimenconditions. Ap value,0.05 was considered statistically significant. Data are expressed as mean6 SEM in the Figures.

To identify trends in experimental data, regression mels based on restricted cubic splines~S-Plus,MATHSOFT, Se-attle, WA! were used. ‘‘Goodness’’ of fit for each trend wadetermined using the multipleR2 statistic, which representthe amount of variability in the response variable~e.g., up-take! that is explained by the fitted variable~e.g., pressure!.A multiple R2 of 1 indicates a perfect relationship betwethe fit and response variables, while a multipleR2 of 0 indi-cates no relationship.

III. EXPERIMENTAL RESULTS

In a companion study~Guzman et al., 2001!, we mea-sured the effects of acoustic pressure, exposure time,pulse length on the uptake of a model compound, calcand the loss of cell viability in DU145 prostate cancer aaortic smooth muscle cell suspensions in the presence oftison contrast agent. As is commonly done, each cell samwas treated as a single homogeneous population, and upand viability were expressed as overall average valuesresentative of each cell sample. However, closer examinaof the data shows that cell samples are heterogeneoustherefore should be described with multiple subpopulati~Fig. 1!. Therefore, in this study the data were reanalyzedaccount for the observed heterogeneity.

A. Cell heterogeneity

Heterogeneous bioeffects were observed in both DUand AoSMC cell samples at almost all of the ultrasouconditions tested. Figure 1 contains histograms of calcfluorescence associated with viable cells~cells rendered nonviable by ultrasound are discussed further in the followsections!. As shown in Fig. 1~a!, the fluorescence emittefrom a representative control sample of viable cells is dtributed across a single population having low fluorescenFigures 1~b!–~d! show a representative set of samplesposed to ultrasound. The cells in these samples have fluocence~i.e., uptake! corresponding to one of three regions:~1!a low-fluorescence peak,~2! a high-fluorescence peak, or~3!a wide valley in between. All samples exposed to ultrasoushowed this distribution containing two peaks and a wvalley. Only the relative heights of the peaks varied amosamples@Figs. 1~b!–~d!#.

The low-fluorescence peak of Figs. 1~b!–~d! contains asubpopulation of cells with fluorescence similar to that oserved in control samples and is termed the nominal up

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population ~NUP!. Although NUP cells have fluorescencsomewhat higher than that of control cells, this level of flurescence is just above the detection limit of the fluorescemeasurement and may correspond to low-level bindingcalcein to cell membranes following ultrasound exposuBecause fluorescence emitted by these cells was so dimwas not possible to visualize by microscopy whetherfluorescence was associated with the cell membranecytosol.

The second and third subpopulations in Figs. 1~b!–~d!contain cells with higher levels of uptake. Those cells in tbroad valley are defined as the low uptake population~LUP!,while those in the highly fluorescent peak are defined ashigh uptake population~HUP!. Although, the histograms inFig. 1 might also be described using just two populations,example, with non-Gaussian distributions, we felt that thpopulations represent the data better, since the broad ranfluorescence found in the ‘‘valleys’’ warrants its own dscriptor ~i.e., LUP!. The three-subpopulation classificatioprovides a means to describe data concisely, that woulderwise be difficult to describe, using the simplest fit that donot leave out important information. This categorization inthree subpopulations is based solely on phenomenologobservation and is not based on theoretical or mechanconsiderations.

As further evidence that cells exposed to the same ulsound conditions can respond with highly heterogeneamounts of uptake, confocal microscopy was used to imcalcein fluorescence within viable cells. Figure 2 shows thadjacent cells that experienced the same ultrasound esure, but have very different fluorescence intensities. Thewith almost no visible fluorescence is representative of NUthe cell with brighter fluorescence represents LUP, andcell with the brightest fluorescence represents HUP.

B. Molecular uptake within each subpopulation

The average number of calcein molecules taken upviable cells within each of the three subpopulations of

FIG. 2. A confocal microscopy image of three adjacent cells showssimultaneous presence of three calcein uptake subpopulations.~A! Thebrightly fluorescent cell is indicative of high uptake of green-fluoresccalcein~HUP!, ~B! the dimmer fluorescent cell is indicative of low uptak~LUP!, and ~C! the dark cell is indicative of nominal uptake~NUP!. Cellnuclei are identified by an asterisk. In this image, fluorescence is due tocombined signals from calcein uptake and Hoescht 33342 nuclear stainNUP cell nucleus is observed by the faint glow given off by the Hoesdye. In the LUP and HUP cells, the nuclei are indicated by the densbright signal that results from combined calcein and Hoescht fluoresce

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sonated cells was determined using flow cytometry.shown in Fig. 3, calcein uptake was significantly differenteach of the three subpopulations~Student’s t-test,p,0.001!.On average, NUP cells took up 4.8(65.9)3104

molecules/DU145 cell and 5.7(67.8)3104 molecules/

FIG. 3. The number of calcein molecules delivered per cell in each spopulation: NUP~white!, LUP ~gray!, and HUP~black!. Ultrasound peaknegative pressures were:s50.6, h51.2, n51.6, L52.0, 352.4,!52.8, and153.0 MPa. Points are connected with solid lines~DU145! ordashed lines~AoSMC!.


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AoSMC cell, LUP cells took up 3.0(61.9)3106 molecules/DU145 cell and 2.2(61.2)3106 molecules/AoSMC cell,and HUP cells took up 1.36(60.5)3107 molecules/DU145 cell and 1.1(60.3)3107 molecules/AoSMC cell(mean6standard error!. Because the fraction of cells withineach subpopulation varied substantially~as discussed in thefollowing section!, overall uptake for a total population ocells ~i.e., the sum of uptake from all three subpopulatioweighted by the fraction of viable cells in each subpopution! exhibits a strong dependence on ultrasound exposconditions, as shown in our companion study~Guzman et al.,2001!.

C. Pressure dependence of subpopulationdistribution among viable cells

Having established that within each subpopulationnumber of molecules per cell showed small variation~Fig.3!, we wanted to determine the effects of ultrasound exsure conditions on the distribution of cells between the thsubpopulations. Figure 4 shows the effect of pressure ondistribution among the viable cells. The NUP fraction of vable cells~NUPviable; white bars! generally decreased withincreasing pressure~statistical analysis provided in Fig. 4!.In contrast, the LUP fraction of viable cells~LUPviable; graybars! remained relatively constant, and the HUP fractionviable cells~HUPviable; black bars! generally increased withincreasing acoustic pressure, indicating that the composiof viable cells became richer in HUP at higher pressures

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FIG. 4. Subpopulation distribution among viable cells as a function of pressure at different exposure times. The white bars indicate cells in NUP,y barsindicate LUP, and black bars indicate HUP. The fraction of cells in NUP decreased, the fraction of cells in LUP varied little, and the fraction of cellsn HUPincreased with increasing peak negative pressure in both~A! DU145 and~B! AoSMC cell samples. Ultrasound peak negative pressures were:a50, b50.6, c51.2, d51.6, e52.0, f 52.4, g52.8, andh53.0 MPa. Pulse length was held constant at 60 ms. One-way ANOVA indicates statistical signifiof dependence on acoustic pressure for the fraction of cells in each sub-population:2(p.0.05), 1 (p,0.05), 11 (p,0.01),111 (p,0.001), for NUP~upper!, LUP ~middle!, and HUP~lower! subpopulations.


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FIG. 5. Subpopulation distributionamong viable cells as a function of exposure time at different pressures. Thwhite bars indicate cells in NUP, graybars indicate LUP, and black bars indicate HUP. The fraction of cells inNUP decreased, the fraction of cells iLUP varied little, and the fraction ofcells in HUP increased with increasinexposure time in both~A! DU145 and~B! AoSMC cell samples. Ultrasoundexposure times werei 50, j 5120, k5240, l 5540, m51000, n52000 ms. Pulse length was held constant at 60 ms. One-way ANOVA in-dicates statistical significance of dependence on exposure time for thfraction of cells in each subpopulation2(p.0.05), 1 (p,0.05), 11 (p,0.01), 111 (p,0.001), for NUP~upper!, LUP ~middle!, and HUP~lower! subpopulations.

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D. Exposure time dependence of subpopulationdistribution among viable cells

A similar trend was observed when studying the effeof ultrasound exposure time at constant pressure. To demstrate this more clearly, the data in Fig. 4 were replotted afunction of exposure time in Fig. 5, which shows thNUPviable generally decreased, LUPviable remained relativelyconstant, and HUPviable generally increased with increasinexposure time. As with increasing pressure, the composiof viable cells became richer in HUP at longer expostimes.


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E. Subpopulation distribution among all cells

The analysis presented so far has addressed onlycells that remain viable after exposure to ultrasound andvided them into three subpopulations. However, large nubers of cells can be made nonviable by ultrasound. A mcomplete analysis should consider that a cell exposed totrasound could have one of four fates: either it remainsable and falls into the NUP, LUP, or HUP subpopulationit is rendered nonviable.

To account for nonviable cells, we recalculated the d

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FIG. 6. Subpopulation distribution among all cells as a function of pressure at different exposure times. The white bars indicate cells in NUP,rsindicate LUP, and black bars indicate HUP. Overall DU145~A! and AoSMC~B! cell viability decreased with increasing pressure at constant exposurePercent cells in NUP decreased with increasing pressure. Percent cells in LUP and HUP changed little as pressure was varied except when viabilerylow. Ultrasound peak negative pressures were:a50, b50.6, c51.2, d51.6, e52.0, f 52.4, g52.8, andh53.0 MPa. Pulse length was held constant atms. One-way ANOVA indicates statistical significance of dependence on acoustic pressure for the fraction of cells in each subpopulation:2 (p.0.05),1 (p,0.05), 11 (p,0.01), 111 (p,0.001), for NUP~upper!, LUP ~middle!, and HUP~lower! subpopulations.


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FIG. 7. Subpopulation distribution among all cells as a function of exposure time at different pressures. The white bars indicate cells in NUP,rsindicate LUP, and black bars indicate HUP. Overall DU145~A! and AoSMC~B! cell viability decreased with increasing exposure time at constant pnegative pressure. Percent cells in NUP decreased with increasing exposure time. Percent cells in LUP and HUP changed little as exposure timeedexcept when viability was very low. Ultrasound exposure times were:i 50, j 5120, k5240, l 5540, m51000, andn52000 ms. Pulse length was helconstant at 60 ms. One-way ANOVA indicates statistical significance of dependence on exposure time for the fraction of cells in each subp2 (p.0.05),1 (p,0.05), 11 (p,0.01), 111 (p,0.001), for NUP~upper!, LUP ~middle!, and HUP~lower! subpopulations.

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tribution of cells among the three subpopulations as a frtion of all cells present during ultrasound exposure. Figureand 7 present the distribution of all cells as functionspressure and exposure time. The height of each bar resents the fraction of cells that remained viable and the whgray, and black bars represent the three subpopulationviable cells. These figures show that, overall, cell viabilgenerally decreased with increasing pressure and expotime, as discussed previously~Guzman et al., 2001!. Figures6 and 7 also demonstrate that increasing pressure and esure time generally caused a decrease in NUPall cells ~i.e., theNUP subpopulation expressed as a fraction of all cellsposed to ultrasound!, but generally did not affect LUPall cells

or HUPall cells. At long exposure times and high pressurviability was extremely low and therefore the LUP and HUsubpopulations were decreased.

These trends contrast with those observed in Figs. 45, which did not account for the loss of viability associatwith increasing pressure and exposure time. The analshown in Figs. 4 and 5 on the basis of only viable cells mbe useful for scenarios where cell death is not of primconcern, but delivery of molecules is critical. For examplaboratory scientists may be more concerned with efficieof gene or protein uptake among viable cells, since theviving cell population can be rapidly grown in cultureyield more cells. Alternatively, analysis on the basis ofcells shown in Figs. 6 and 7 may be useful for medicalsearchers and clinicians interested in ultrasound conditthat deliver large amounts of therapeutic material withexcessive cell death~e.g., useful for targeted drug delivery ogene therapy! or with extensive cell death~e.g., useful forcancer treatment!.


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F. Pulse length dependence of subpopulationdistribution

When pulse length was varied between 20ms ~10 cycles/pulse! and 60 ms~30 000 cycles/pulse!, subpopulation distri-butions did not change significantly~Fig. 8!. This conclusionis similar to the result presented previously~Guzman et al.,2001!, where overall viability was not affected by varyinpulse length over the range of conditions tested.

FIG. 8. Subpopulation distribution among all cells as a function of pulength. The distribution of subpopulations does not vary significantly wpulse length over the range of 0.02–60 ms at 240-ms exposure time1.6-MPa peak negative acoustic pressure. Black bars indicate cells in Hgray bars indicate LUP, and white bars indicate NUP. One-way ANOindicates statistical significance of dependence on pulse length for thetion of cells in each subpopulation:p value50.53, 0.87, and 0.79 for NUPLUP, and HUP cells, respectively.


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FIG. 9. Subpopulation distribution among viable cells as a functionacoustic energy exposure.~A! The NUP subpopulation decreased~multipleR250.68 and 0.59 for DU145 and AoSMC cells, respectively!, ~B! the LUPsubpopulation varied little~multiple R250.13 and 0.29 for DU145 andAoSMC cells, respectively!, and~C! the HUP subpopulation increased witincreasing energy~multiple R250.66 for both DU145 and AoSMC cells!~s5DU145, d5AoSMC!.


G. Acoustic energy correlation with subpopulationdistribution

Because the distribution of cell subpopulations depestrongly on exposure time and even more strongly on p

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FIG. 10. Subpopulation distribution among all cells as a function of acouenergy exposure.~A! The NUP subpopulation decreased~multiple R2

50.79 and 0.76 for DU145 and AoSMC cells, respectively! and ~B! LUPand~C! HUP subpopulations increased, reached a maximum, and decrewith increasing energy~LUP: multiple R250.41 and 0.56 for DU145 andAoSMC cells; HUP: multipleR250.27 and 0.42 for DU145 and AoSMCcells! ~s5DU145, d5AoSMC!.


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sure, we hypothesized that the families of distributioshown in Figs. 4–8 could be collapsed down into sincurves when plotted as a function of acoustic energy exsure ~E!. In Fig. 9, the subpopulation distributions amonviable cells~Figs. 4 and 5! were replotted as a function oacoustic energy exposure. Figure 9 shows that for bothtypes the data collapsed into single curves, where NUPviable

decreased, LUPviable was scattered, and HUPviable increasedwith increasing acoustic energy exposure.

The subpopulation distributions calculated on the baof all cells were also correlated with acoustic energy exsure. For this, the data from Figs. 6 and 7 were replotversus energy in Fig. 10. The figure shows that NUPall cells

decreased (ANOVAp,0.05); LUPall cells and HUPall cells

probably initially increased~since no LUP or HUP cells werepresent in controls!, leveled out (E,50 J/cm2, ANOVA p.0.05), and then decreased (E.50 J/cm2, ANOVA p,0.05) with increasing acoustic energy exposure.

H. Acoustic energy correlation with molecular uptake

Figure 3 indicates that uptake within each subpopulat~i.e., NUP, LUP, and HUP! was not significantly differentfrom each other. This is shown again in Fig. 11, in whithese data are replotted versus acoustic energy expoHowever, the scatter within each subpopulation can betially explained by an increasing trend with increasing eergy for NUP and LUP cells~one-way ANOVAp,0.01!.Uptake within the HUP subpopulation was independentenergy exposure~one-way ANOVAp.0.10!.

The average uptake in the HUP subpopulation~i.e., av-erage of all HUP data points in Fig. 11! was 1.36 (60.5)3107 and 1.10(60.3)3107 molecules per DU145 andAoSMC cell, respectively. Based on average cell volumes2200mm3 for DU145 cells and 2400mm3 for AoSMC cells~Guzman et al., 2001!, this corresponds to intracellular concentrations of 10.363.7 and 7.662.1mM, respectively. Be-cause calcein was supplied extracellularly at 10mM, averageuptake by HUP cells approached the maximum possbased on chemical equilibrium in the absence of binding

IV. DISCUSSION

A. Cavitation-based versus cell-based heterogeneity

The most notable finding in this study is that cells eposed to the same acoustic environment exhibited cell-to-heterogeneity in their response to ultrasound, which isdirect support of our first hypothesis. This observation raian interesting question: Are these heterogeneous effectsto ~i! spatial and temporal heterogeneity in cavitation genated by ultrasound or~ii ! heterogeneity based on differencbetween cells and their responses to acoustic cavitation?

In support of the first explanation, cavitation is knownbe a stochastic phenomenon controlled by the locationavailability of nucleation sites, which makes cavitation etremely heterogeneous in both time and space~Leighton,1994; Miller et al., 1996; Barnett, 1998!. As a result, a cell’sfate may be determined by the degree to which that cell cain contact with one or more of the finite number of stableinertial cavitation bubbles. The mechanism of cell disrupt


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could involve just a single cell-with-bubble interaction or thaccumulated effect of multiple interactions. In this way, cethat do not come into sufficiently close contact with a catation bubble experience no effect~i.e., NUP!; cells that haveprogressively closer interactions with bubbles fall amongbroad LUP and HUP subpopulations; and cells that locaexperience too much cavitation are made nonviable. Baon the calculation method described by Wardet al. ~2000!the average initial cell-to-bubble spacing in the present stwas 45mm, which is 2–3 times the diameter of the ceused.

Heterogeneity in cavitation could also come from insuficient mixing. The literature indicates that exposure vesrotation may be necessary to promote cell–bubble mixand thereby enhance bioeffects~Churchet al., 1982!. How-ever, other studies have shown that when contrast anucleation sites are present, there is no difference in biofects caused in rotating and nonrotating exposure ves~Miller et al., 1999; Brayman and Miller, 1999!. For our ex-periments, in which contrast agent was present, we visuobserved vigorous mixing within cell samples during ultrsound exposure and therefore believe that insufficient mixwas not the source of heterogeneity. Instead the finite nber and short lifetime of bubbles may limit the odds of haing cell–bubble interactions.

The second possible explanation for heterogeneitybased on biological differences between individual cells taffect the cell’s mechanical properties or otherwise influenhow each cell interacts with cavitation bubbles. In this caall cells could have identical cell–bubble interactions~i.e.,no time-averaged heterogeneity in cavitation!, but differentsubpopulations of cells might react to cavitation differentBased on differences in cell cycle or other factors, some c

FIG. 11. The average number of calcein molecules delivered per cell~com-bined plot of DU145 and AoSMC cells! for each uptake subpopulation asfunction of acoustic energy exposure. Calcein uptake per cell increasedfunction of energy exposure for NUP and LUP cells, but showed no depdence for HUP cells.


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might be reversibly affected by a given ultrasound expos~i.e., LUP or HUP!, others might be unaffected~i.e., NUP!,and still others might be rendered nonviable. Additional eperiments are needed to determine the relative roles oferogeneity in cavitation and between cells as the causheterogeneity in uptake and cell viability.

B. Comparison to heterogeneity and homogeneityseen in other studies

Heterogeneity and homogeneity seen in other studiesing ultrasound and a related phenomenon, electroporaprovide additional perspective on the results reported hPrevious ultrasound studies have observed heterogeneithe form of three possible responses: cells that are ufected, induced to take up molecules, or rendered nonviaFor example, the literature has numerous reports of ulsound having heterogeneous effects on cell viability, whultrasound renders only a fraction of cells nonviable~Bray-man and Miller, 1999; Churchet al., 1982; Greenleafet al.,1998; Miller et al., 1996!. Other studies have reported heerogeneity in gene transfection, which is an indirect measof the combined effects of gene uptake and cell viabi~Bao et al., 1997; Greenleafet al., 1998!. While the presentstudy confirms and quantifies these earlier observationintroduces an additional aspect of heterogeneity: among cinduced to take up molecules, there is a broad distributiolevels of molecular uptake, that starts just above control vues ~NUP! and approaches chemical equilibrium with textracellular solution~HUP!. This result has not been reported before, probably because the cell-by-cell measments needed to show it have not been performed.

Ultrasound’s heterogeneous effects are dramaticallyferent from the distribution of molecular uptake causedelectroporation. Electroporation is a phenomenon that slarly causes varying degrees of molecular uptake andviability in response to a brief~e.g.,ms to ms! electric fieldpulse~Changet al., 1992!. In contrast to results seen here,cells subjected to a given electroporation exposure have bshown to take up molecules in a homogeneous mannestudies with DU145 cells~Canatellaet al., 2001! and humanred blood cell ghosts~Prausnitzet al., 1993!. For sphericalcells, electroporation histograms similar to those in Fig. 1not show multiple populations, but are always characteriby a single peak with an approximately Gaussian distrition. Moreover, the location of that peak can occur anywhalong thex axis of the histogram, depending on electropotion conditions used. This difference may be explainedthe spatial and temporal uniformity of the electric field duing electroporation, as opposed to the heterogeneity of ctation.

C. Significance of correlation with acoustic energyexposure

In support of our second hypothesis, heterogeneitymolecular uptake was found to correlate with acousticergy exposure. A similar dependence on energy expowas shown previously for cell viability and total moleculuptake~Guzman et al., 2001! and increased skin conductiv


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ity ~Mitragotri et al., 2000!. This dependence on energyimportant because it provides a single parameter that colates with the various bioeffects induced by extremely coplex cavitational activity. Using this correlation, the oserved bioeffects can be approximately described by eneexposure regardless of the applied pressure, exposureor pulse length. This approach may allow a researcheclinician to identify ultrasound conditions that yield usefeffects. For example, a physician performing gene thermay want to maximize HUPall cells and therefore apply theoptimal energy exposure~e.g., 50–350 J/cm2 in the presentstudy, see Fig. 10! to deliver large numbers of moleculenecessary for transfection into as many cells as possible.targeted chemotherapy, one may want to minimNUPall cells and therefore operate at a much higher energyassure that all cancer cells are killed either directly by ultsound or indirectly due to uptake of a chemotherapeuagent.

V. CONCLUSIONS

When measured on a cell-by-cell basis, the numbermolecules taken up by cells exposed to the same ultrasoconditions was shown to be extremely heterogeneous. Tcell-to-cell heterogeneity was observed in both AoSMC aDU145 cells at all of the ultrasound conditions tested. Afexposure to ultrasound, viable cells could be divided inthree subpopulations, for which the fraction of cells in easubpopulation depended strongly on pressure and expotime, but not on pulse length, and was found to correlate wacoustic energy exposure. Molecular uptake within each spopulation remained relatively constant. Because ulsound’s effects are thought to be mediated by cavitation,existence of subpopulations suggests heterogeneity intime and location of acoustic cavitation and/or in celluresponses to cavitation.

ACKNOWLEDGMENTS

We would like to thank Dr. Paul Canatella, Dr. KeyvaKeyhani, Dr. Thomas Lewis, Dr. Russel Heikes, and Dr. Pter MacDonald for their helpful comments and suggestio

Bao, S., Thrall, B. D., and Miller, D. L.~1997!. ‘‘Transfection of a reporterplasmid into cultured cells by sonoporationin vitro,’’ Ultrasound Med.Biol. 23, 953–959.

Barnett, S.~1998!. ‘‘Nonthermal issues: cavitation—its nature, detectioand measurement,’’ Ultrasound Med. Biol.24, S11–S21.

Brayman, A. A., and Miller, M. W. ~1999!. ‘‘Sonolysis of Albunex-supplemented, 40% hematocrit human erythrocytes by pulsed 1-MHztrasound: Pulse number, pulse duration, and exposure vessel rotatiopendence,’’ Ultrasound Med. Biol.25, 307–314.

Canatella, P. J., Karr, J. F., Petros, J. A., and Prausnitz, M. R.~2001!.‘‘Quantitative study of electroporation-mediated molecular uptake andviability,’’ Biophys. J. 80, 755–764.

Chang, D. C., Chassy, B. M., Saunders, J. A., and Sowers, A. E.,~1992!.Guide to Electroporation and Electrofusion~Academic, New York!.

Church, F. C., Flynn, H. G., Miller, M. W., and Sacks, P. G.~1982!. ‘‘Theexposure vessel as a factor in ultrasonically-induced mammalian cell lII,’’ Ultrasound Med. Biol.8, 299–309.

Fechheimer, M., Denny, C., Murphy, R. F., and Taylor, D. L.~1986!.‘‘Measurement of cytoplasmicpH in dictyostelium discoideum by using anew method for introducing macromolecules into living cells,’’ Eur.Cell Biol. 40, 242–247.


-es.

nd

o

tra

r-

, R.ed

nda

Gift, E. A., and Weaver, J. C.~1995!. ‘‘Observation of extremely heterogeneous electroporative molecular uptake bySaccharomyces cerevisiawhich changes with electric field pulse amplitude,’’ Biochim. BiophyActa 1234, 52–62.

Greenleaf, W. J., Bolander, M. E., Sarkar, G., Goldring, M. B., aGreenleaf, J. F.~1998!. ‘‘Artificial cavitation nuclei significantly enhanceacoustically induced cell transfection,’’ Ultrasound Med. Biol.24, 587–595.

Guzman, H. R., Nguyen, D. X., Sohail, K., and Prausnitz, M. R.~2001!.‘‘Ultrasound-mediated disruption of cell membranes. I. Quantificationmolecular uptake and cell viability,’’ J. Acoust. Soc. Am.110, 588–596.

Harrison, G. H., Balcer-Kubiczek, E. K., and Gutierrez, P. L.~1996!. ‘‘ Invitro mechanisms of chemopotentiation by tone-burst ultrasound,’’ Ulsound Med. Biol.22, 355–362.

Leighton T. G.~1994!. The Acoustic Bubble~Academic, New York!.Miller, D. L., Bao, S., and Morris, J. E.~1999!. ‘‘Sonoporation of cultured

cells in the rotating tube exposure system,’’ Ultrasound Med. Biol.25,143–149.


f

-

Miller, M. W., Miller, D. L., and Brayman, A. A.~1996!. ‘‘A review of invitro bioeffects of inertial ultrasonic cavitation from a mechanistic pespective,’’ Ultrasound Med. Biol.22, 1131–54.

Mitragotri, S., Farrell, J., Tang, H., Terahara, T., Kost, J., and Langer~2000!. ‘‘Determination of threshold energy dose for ultrasound-inductransdermal drug transport,’’ J. Controlled Release63, 41–52.

Prausnitz, M. R., Lau, B. S., Milano, C. D., Conner, S., Langer, R., aWeaver, J. C.~1993!. ‘‘A quantitative study of electroporation showingplateau in net molecular transport,’’ Biophys. J.65, 414–422.

Robinson, J. P., and Kelley, S., computer codeLDATA , Purdue University,West Lafayette, IN, 1998.

Saad, A. H., and Hahn, G. M.~1987!. ‘‘Ultrasound enhances Adriamycintoxicity in vitro,’’ in Heat Transfer in Bioengineering and Medicine, ed-ited by J. C. Chato, T. E. Diller, K. R. Diller, and R. B. Roemer~Ameri-can Society of Mechanical Engineers Press, New York!.

Ward, M., Wu, J., and Chiu, J.~2000!. ‘‘Experimental study of the effects ofOptison concentration on sonoporationin vitro,’’ Ultrasound Med. Biol.26, 1169–1175.


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