PII S0360-3016(98)00311-3

● Chemical Sensitizers and Protectors

ELECTROPORATION INCREASES THE EFFECT OF BOROCAPTATE(10B-BSH) IN NEUTRON CAPTURE THERAPY

KOJI ONO, M.D., PH.D., YUKO KINASHI, M.D., PH.D., SHIN-ICHIRO MASUNAGA, M.D., PH.D.,MINORU SUZUKI, M.D., AND MASAO TAKAGAKI , M.D., PH.D.

Radiation Oncology Research Laboratory, Research Reactor Institute, Kyoto University, Noda, Kumatori-cho, Sennan-gun,Osaka, Japan

Purpose: The cell membrane permeability of borocaptate (10B-BSH) and its extent of accumulation in cells arecontroversial. This study was performed to elucidate these points.Methods and Materials: Two different treatments were applied to SCCVII tumor cells. The first group of tumorcells was incubated in culture medium with10B-BSH or 10B-enriched boric acid, and was exposed to neutronsfrom the heavy water facility of the Kyoto University Reactor (KUR). More than 99% of neutrons were thermalneutrons at flux base. The second group was pretreated by electroporation in combination with10B-BSH, andthereafter the cells were irradiated with neutrons. The cell killing effects of boron neutron capture therapy(BNCT) using BSH were investigated by colony formation assay.Results: Surviving cell fraction decreased exponentially with neutron fluence, and addition of BSH significantlyenhanced the cell killing effect of neutron capture therapy (NCT) depending on10B concentration. The effect ofBSH-BNCT also increased with preincubation time of cells in the medium containing BSH. The electroporationof cells with BSH at 10 ppm10B markedly enhanced BSH-BNCT effects in comparison with that of preincubationalone. The effect of BSH-BNCT with electroporation was equal to that of BNCT using10B-boric acid at a same10B concentration (10 ppm).Conclusions: BSH is suggested to penetrate the cells slowly and remained after washing. Electroporation canintroduce BSH into the cells very efficiently, and BSH stays in the cells and is not lost by washing. Therefore, ifelectroporation is applied to tumors after BSH injection, 10B remains in tumors but is cleared from normaltissues, and selective accumulation of10B in tumors will be achieved after an adequate waiting time. © 1998Elsevier Science Inc.

BSH, Electroporation, SCCVII tumor cells.

INTRODUCTION

10B can capture thermal neutrons at an extremely highprobability (3837 barn) in comparison with1H, 12C, 14N,and16O, and release an alpha particle and a recoiling7Li ionwith an average total kinetic energy of 2.34 MeV,10B(n,a)7Li. These particles (a, 7Li1) are high LET radia-tion with very limited tracks which do not exceed 1 celldiameter (10mm) (1). Therefore, if10B accumulates in tumorcells selectively, the cells can be destroyed completely withminimal effects on adjacent cells containing no10B. 10B-enriched borocaptate sodium (BSH: Na2B12H11SH) hasbeen used as an agent for boron neutron capture therapy(BNCT) of malignant glioma (2). BSH does not cross theblood–brain barrier (BBB) into the normal brain, but accu-mulates in malignant brain tumors because of their defectiveBBB. However, the membrane permeability of BSH and

BSH accumulation in tumor cells are controversial. More-over in tumors in other organs, BSH distributions are notselective because of the lack of an appropriate selectivebarrier such as the BBB (3). Because the tracks of alphaparticles and recoiling7Li ions are very short, the energydeposition in DNA varies depending upon the site whereboron neutron capture reaction occurs. This difference ofreaction site greatly affects cell killing as detected by acolony formation assay. Therefore, if there was a way toinject BSH into cells the above controversy could be ad-dressed by comparing the cell killing effects of BSH-BNCTcombined with and without the approach. It has been re-ported that passage of an electric current across a cellmembrane can increase its permeability, and this technique(known as electroporation) has frequently been applied tointroduce drugs into cells (4). In this study electroporationwas employed to introduce BSH into cells, and the signif-

Presented at the 10th International Conference on ChemicalModifiers of Cancer Treatment, Clearwater, FL, Jan 28–31, 1998.

Reprint requests to: Koji Ono, M.D., Ph.D., Radiation OncologyResearch Laboratory, Research Reactor Institute, Kyoto Univer-sity, Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan.

Acknowledgments—The authors thank Mrs. Syoko Ono for tech-nical assistance. This study was supported by Grants-in-Aid forCancer Research from the Ministry of Education, Science andCulture of Japan (09255228, 09470201).

Accepted for publication 6 July 1998.

Int. J. Radiation Oncology Biol. Phys., Vol. 42, No. 4, pp. 823–826, 1998Copyright © 1998 Elsevier Science Inc.Printed in the USA. All rights reserved

0360-3016/98/$–see front matter

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icance of the combination of BSH and electroporation as anapproach for improving BNCT was investigated.

METHODS AND MATERIALS

Cell line and BSHSCCVII tumor cells, exponentially growing in Eagle’s

minimum essential medium supplemented with 292 mg/LL-glutamine and 12.5% fetal calf serum, were trypsinizedand single-cell suspensions in phosphate-buffered saline orcomplete medium were prepared. BSH was purchased fromBBI (Boron Biologicals, Inc., Raleigh, NC). BSH-dissolvedsaline solution, about 400 ppm at10B base, was made andsterilized by filtering. An exact10B concentration of theBSH solution was measured by prompt-g-ray spectrometry(5). The BSH solution was added into tumor cell suspen-sions containing 50–1003 104 cells per ml at a final10B-concentration of 10 and 20 ppm.

ElectroporationTumor cell suspensions containing BSH were put into the

chamber, maximum effective volume of 0.8 ml, of theBio-Rad Laboratories Gene Pulser for electroporation. Elec-troporation was performed under the following conditions:electrical field strength5 0.75 kV/cm (actual voltage5 0.3kV/0.4 cm electrode gap), capacitance5 960mF. Treatmenttime varied from 10 to 12 sec. The demolition of 80–90%of cells was noticed by phase contrast microscope followingelectroporation.

Neutron irradiation and colony formation assayAfter electroporation, the cell suspension was centri-

fuged, resuspended in fresh complete medium, and 2 ml ofcell suspension was put into a small Teflon tube for thermalneutron irradiation. As a control experiment, the tumor cellsuspension with BSH solution was incubated for 2 or 16hours, and thereafter the suspension was centrifuged toremove BSH, resuspended in fresh complete medium, andreceived thermal neutron irradiation as the cells followingan electroporation did. To examine the extent of ability ofelectroporation to allow BSH to enter cells, the cells sus-pended in complete medium containing10B-enriched boricacid were irradiated with neutrons. The neutron beam witha cadmium ratio of 148, i.e., a mixture beam of 23 109

thermal, 1.43 107 epithermal, and 2.83 106 fast neutronsat flux base (cm22s21), was used in this study. The neutronfluence was measured by the spectrometry ofg-rays from198Au produced in the reaction of197Au(n,g)198Au as wedid in a previous study (6). Au foils were placed on both thesides, front and back of the Teflon tube, and the fluence ofneutrons that reached the cells in the Teflon tube wasrepresented by an arithmetic mean value. After irradiation,an appropriate number of tumor cells to form 50 to 100colonies were inoculated onto 60-mm-diameter Petri dishescontaining complete medium. After incubation for 10 days,the colonies were fixed with ethanol, stained with crystalviolet, and counted by the aid of low-power microscope.

The plating efficiency (PE) of the cells without electropo-ration nor the cells that escaped destruction by electropora-tion was 84.56 6.8%. The surviving cell fraction (SF) wasobtained by dividing the PE of the cells that receivedneutrons by that without neutrons. The best fitted linesbetween neutron fluence and SF were determined by theleast squares method. The difference between slopes of cellsurvival curves was statistically analyzed.

RESULTS AND DISCUSSION

The surviving cell fraction decreased exponentially withneutron fluence, and addition of BSH significantly enhancedthe cell killing effect of neutron capture therapy (NCT) in a10B concentration-dependent manner, i.e., the slopes of cellsurvival curves were 0.254 (60.0105)3 10212cm2s, 0.427(60.0369) 3 10212cm2s, and 0.525 (60.0116) 310212cm2s for neutrons alone, for 10 ppm10B 2-hr incuba-tion, and for 20 ppm10B 2-hr incubation, respectively (Fig.1 and Table 1). BNCT effects also significantly increasedwith preincubation time, i.e., cell survival curve slopes for16-hr incubation were 0.675 (60.0735)3 10212cm2s and0.802 (60.0496)3 10212cm2s at 10 ppm and 20 ppm10B,respectively (Fig. 1 and Table 1). A report states that BSH-BNCT effect was almost completely lost after washing cellseven after 24-hr preincubation (7), however our studyshowed different results, i.e., the BNCT effects increasedwith preincubation time of cells with BSH and remained

Fig. 1. Effect of10B concentration and preincubation time on cellsurvival curves following BNCT.Open triangles:BSH (10B 10ppm)–2-hr incubation–wash–neutrons.Filled triangles:BSH (10B10 ppm)–16-hr incubation–wash–neutrons.Open squares:BSH(10B 20 ppm)–2-hr incubation–wash–neutrons.Filled squares:BSH (10B 20 ppm)–16-hr incubation–wash–neutrons.Open cir-cles: neutron irradiation alone.

824 I. J. Radiation Oncology● Biology ● Physics Volume 42, Number 4, 1998

after washing. These findings suggest that BSH slowlypenetrates cells and remains after washing.

The electroporation alone did not increase the sensitivityof cells to neutrons, i.e., slopes of cell survival curves were0.254 (60.0105) 3 10212cm2s and 0.273 (60.0125) 310212cm2s for neutron alone and for an addition of electro-poration without BSH, respectively (Fig. 2 and Table 2).This indicates that the electroporation does not always de-stroy the radiation-resistant phase cells. On the other hand,the electroporation of cells with BSH at 10 ppm10B mark-edly enhanced BSH-BNCT effect in comparison with thatof preincubation alone. The slopes of cell survival curves

were 0.990 (60.100)3 10212cm2s and 0.427 (60.0369)310212cm2s for BSH plus electroporation and for BSH with-out electroporation, respectively (Fig. 2 and Table 2). Theeffect of BSH-BNCT with electroporation was equal to thatof BNCT using10B-boric acid at the same10B concentration(10 ppm) (Fig. 2 and Table 2).10B-boric acid is consideredto enter the cells freely, i.e., an intracellular10B concentra-tion can reach the same level as in the medium, and itshowed a marked enhancement effect of NCT as seen inFig. 2. These data indicate that electroporation can intro-duce BSH into the cells very efficiently, and BSH stays inthe cells and is very hard to wash out. The BSH-BNCTeffect at 10 ppm10B with electroporation was significantlylarger than that at 10 ppm10B for 16-hr preincubation (p ,0.05), and it was also larger than that at 20 ppm10B for16-hr preincubation although its difference is not signifi-cant. An enhancement ratio of over 2 by electroporation isexpected from these data. After administration of BSH tocancer patients, BSH clears from blood with aT1/2 of 6–7hours (8). Therefore, if electroporation was applied to tu-mors after BSH injection,10B would remain in tumors butbe cleared from normal tissues, and selective accumulationof 10B in tumors would be achieved after an adequatewaiting time.

The cytotoxicity of bleomycin is quite low against ade-nocarcinoma cells, and one of the causes of this resistancemight be a cellular membrane block to the entry of the druginto the cells. When bleomycin was combined with electro-poration, however, the cytotoxicity against gastric cancercells in vitro markedly increased (9). This is attributable toan increased cell membrane permeability for bleomycininduced by electroporation. The same effect was alsoachieved inin vivo tumors transplanted in nude mice (9).Although electroporation is very effective in increasing cellmembrane permeability, direct application of electric cur-rent to tumors situated in the body can produce unaccept-able side effects and many difficulties remain before clinicaluse. As an alternative to electroporation, shock waves wereappliedin vitro to cultured cells andin vivo to tumors, andenhanced drug toxicity was also observed (10). Shockwaves can be focused to lesions deeply situated in the body.Therefore, BSH-BNCT in combination with focused shockwaves may be applicable to cancers other than malignantbrain tumors. Research on BSH-BNCT in combination withfocused shock waves on SCCVII tumor cells and onin vivotumors is under study.

Fig. 2. Effect of electroporation on the cell killing by neutronirradiation with or without boron compounds.Open triangles:BSH (10B 10 ppm)–2-hr incubation–wash–neutrons.Filledsquares: BSH (10B 10 ppm)–electroporation–wash–neutrons.Open circles:neutron irradiation alone.Filled circles: electropo-ration–wash–neutrons, (1): 10B-enriched boric acid (10B 10 ppm-)–neutrons. The data of neutron irradiation alone and BSH (10B 10ppm)–2-hr incubation–wash–neutrons in Fig. 1 were plotted againto elucidate the effect of electroporation.

Table 1. Effect of10B concentration and preincubation time on cell survival curves following BNCT

Treatment groups2lnSF 5 C 1 af,

Cf: Neutron fluence

a(310212)

Neutron alone 20.00115 0.2546 0.0105 p , 0.005BSH(10B,10 ppm)-2 hr-Wash–Neutron 20.0609 0.4276 0.0369 p , 0.05BSH(10B,20 ppm)-2 hr-Wash–Neutron 20.0198 0.5256 0.0116

p , 0.002BSH(10B,10 ppm)-16 hr-Wash–Neutron 20.0924 0.6756 0.0735BSH(10B,20 ppm)-16 hr-Wash–Neutron 0.0360 0.8026 0.0496 NS

NS: not significant.

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82510th Chemical Modifiers Conference● Electroporation increases BSH-BNCT effects● K. ONO et al.

REFERENCES

1. Barth RF, Soloway AH, Fairchild RG. Boron neutron capturetherapy of cancer.Cancer Res1990;50:1061–1070.

2. Hatanaka H. Boron neutron capture therapy for tumors. In:Karim ABMF, Law E, editors. Glioma. Berlin: Springer Ver-lag; 1991. p. 233–249.

3. Wazer DE, Zamenhof RG, Harling OK, Madoc-Jones H.Boron neutron capture therapy. In: March PM, Loeffler JS,editors. Radiation oncology technology and biology. Philadel-phia: W. B. Saunders; 1994. p. 167–191.

4. Zimmermann U, Vienken J, Pilwat G. Development of drugcarrier systems: Electric field induced effects in cell mem-branes.J Electroanal Chem1980;116:553–574.

5. Kobayashi T, and Kanda K. Microanalysis system of ppm-order10B concentrations in tissue for neutron capture therapyby prompt gamma-ray spectrometry.Nucl Instrum Meth1983;204:525–531.

6. Ono K, Masunaga S, Kinashi Y, Takagaki M, Akaboshi M,Kobayashi T, Akuta K. Radiobiological evidence suggestingheterogeneous microdistribution of boron compounds in tu-mors: Its relation to quiescent cell population and tumor cure

in neutron capture therapy.Int J Radiat Oncol Biol Phys1996;34(5):1081–1086.

7. Fairchild RG, Kahl SB, Laster BH, Kalef-Ezra J, Popenoe EA.In vitro determination of uptake, retention, distribution, bio-logical efficacy, and toxicity of boronated compounds forneutron capture therapy: A comparison of porphyrins withsulfhydryl boron hydrides.Cancer Res1990;50:4860–4865.

8. Takagaki M, Oda Y, Miyatake S, Kikuchi H, Osawa M, OnoK, Mori K. Boron neutron capture therapy—Preliminary studyof BNCT with sodium borocaptate (Na2B12H11SH) on glio-blastoma.J Neuro-Oncol1997;35:177–185.

9. Kambe M, Arita D, Kikuchi H, Funato T, Tezuka F, Gamo M,Murakawa Y, Kanemaru R. Enhancement of the efficiency ofanticancer drugs with electroporation: Successful electroche-motherapy against gastric cancer cell line in vivo and in vitro.Int J Clin Oncol1997;2:111–117.

10. Kambe M, Ioritani N, Shirai S, Kambe K, Kuwahara M, AritaD, Funato T, Shimodaira H, Gamo M, Orikasa S, KanemaruR. Enhancement of chemotherapeutic effects with focusedshock wave: Extracorporeal shock wave chemotherapy(ESWC).In Vivo 1996;10:369–376.

Table 2. Effect of electroporation on the cell survival curves following neutron irradiation with or without boron compounds

Treatment groups2lnSF 5 C 1 af,

Cf: Neutron fluence

a(310212)

Neutron alone 20.00115 0.2546 0.0105 NSEP(1)-Neutron 20.0745 0.2736 0.0125 p , 0.05BSH(10B,10 ppm)-2 hr-Wash–Neutron 20.0609 0.4276 0.0369 p , 0.001BSH(10B,10 ppm)-EP(1)-Wash–Neutron 0.2070 0.9906 0.100

NSBoric acid (10B,10 ppm)-2 hr–Neutron 20.0670 1.0506 0.108

EP(1): Electroporation was applied.

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