neutron capture autoradiographic determination of 10b distributions and concentrations in biological...

Nuclear Instruments and Methods in Physics Research A 424 (1999) 122—128

Neutron capture autoradiographic determination of10B distributions and concentrations in biological samples for

boron neutron capture therapy

Hironobu Yanagie!,*, Koichi Ogura", Toshio Matsumoto#,Masazumi Eriguchi!, Hisao Kobayashi$

! Department of Surgery, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108, Japan" Physical Science Laboratories, College of Industrial Technology, Nihon University, 1-2-1 Izumi-cho, Narashino, Chiba 275, Japan

# Metabolism and Pharmacokinetics group, Omiya Research Laboratory, Nikken Chemicals Co. Ltd., 1-346 Kitabukuro-cho,Omiya, Saitama 33, Japan

$ Institute for Atomic Energy, Rikkyo University, 2-5-1 Nagasaka, Yokosuka, Kanagawa, 240-01, Japan

Abstract

It is necessary for effective boron neutron capture therapy (BNCT) to accumulate 10B atoms in the tumor cells. Weprepared a cationic liposome entrapped 10B compound for the delivery system and examined the delivery capacity of10B atoms to pancreatic cancer cell, AsPC-1, in vivo. It is required to achieve an accurate measurement of 10B distribu-tions and concentrations in biological samples with a sensitivity in the ppm range for BNCT. We applied CR-39(polyallyldiglycol carbonate) plastic track detectors to a-autoradiographic measurements of the 10B biodistribution insliced whole-body samples of mice. To selectively desensitize undesirable proton tracks, we applied PEW(KOH#C

2H

5OH#H

2O) solution to the etching of CR-39 detector. The subsequent use of an alpha-track radio-

graphic image analysis system enabled a discrimination between alpha tracks and recoiled proton tracks by the track sizeselection method. This enabled us to estimate quantitatively the distributions of 10B concentrations within the tissuesections by comparing with suitable standards. ( 1999 Elsevier Science B.V. All rights reserved.

Keywords: Boron neutron capture therapy; Cationic liposome; Neutron capture a autoradiography; Solid-state nucleartrack detector; CR-39; Desensitization of proton tracks

1. Introduction

The cytotoxic effect of boron neutron capturetherapy (BNCT) is caused by a nuclear reaction

*Corresponding author. Tel.: #81-3-5449-5352; fax: #81-3-5449-5439; e-mail: [email protected].

between 10B and thermal neutrons. This nuclearreaction is as follows:

10B#1nP7Li#4He (a)#2.79 MeV (6.3%)

P7Li*#4He (a)#2.31 MeV (93.7%)

P7Li#4He (a)#0.478 MeV.

0168-9002/99/$ — see front matter ( 1999 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 8 - 9 0 0 2 ( 9 8 ) 0 1 2 8 4 - 4

The resultant 7Li particles and a particles are highlinear energy transfer (LET) particles with relative-ly high biological efficiency. These particles (a and7Li) destroy cells within about 10 lm path lengthfrom the site of capture reaction. It is necessary toaccumulate 10B compounds selectively in tumorcells for selective cyototoxicity of tumor cells with-out affecting adjacent healthy cells.

Liposomes have been investigated extensively ascarriers for anticancer drugs in attempts to directactive agents to tumors or to protect sensitive tis-sues form toxicity [1]. Liposomes are useful drugcarriers, and its is possible to carry a large amountof 10B compound in a liposome, which can bedelivered to tumor cells. We have reported that10B atoms delivered by immunoliposomes arecytotoxic to human pancreatic carcinoma cells(AsPC-1) with thermal neutron irradiation in vitro[2]. The intratumoral injection of boronated im-munoliposomes can increase the retention of10B atoms in tumor cells, causing tumor growthsuppression in vivo under thermal neutron irradia-tion [3].

Multilamellar vesical immunoliposomes are eas-ily phagocytized by the reticuloendothelial system(RES), so it is very difficult to accumulate theliposomal contents in the target cancers in vivo.Forssen et al. have reported that small unilammelarvesicles consisting of highly purified distearoylphosphatidylcholine and cholesterol can producea 10-fold increased delivery of daunorubicin tosolid tumors in vivo [4]. Recently, positivelycharged cationic liposomes were also shown toefficiently introduce genes into cells by formingcomplexes with the plasmid DNA in solution, fa-cilitating fusion of the liposome/DNA complexwith the cell membrane. Cationic liposomes arecomposed of positively charged lipid bilayers, sothey can be bound easily to tumor cells which haveacquired a net negative charge [5].

The accurate measurement of 10B distributionsin biological samples with a sensitivity in the ppmrange is essential for evaluating the potential use-fulness of various boron-containing compounds forBNCT. In this study, we prepared the cationicliposomes as the effective 10B carrier. To test thevalidity, we employed a technique of neutron cap-ture autoradiography using track etch detectors

(SSNTDs). The 10B biodistribution was qualita-tively and quantitatively determined in whole-bodysamples of mice. In the alpha-track etch radio-graphic analysis, alpha tracks were discriminatedfrom proton tracks by carefully selecting the etch-ing solution and the etching condition. The distri-bution and concentration of 10B within the tissuesections were measured by a microscopic analyzingsystem comparing with the results using standardsamples.

2. Materials and methods

2.1. Preparation of target tumor cells

A human pancreatic carcinoma cell line (AsPC-1:Dainihon Seiyaku Co.Ltd.) produces carcinoem-bryonic antigen (CEA) as follows: The AsPC-1 wasmaintained in a medium (PRMI 1640: HazeletonBiologics Inc.) supplemented with 10% fetal calfserum (Cell Culture Labs.) and 100 lg ml~1

kanamycin. All cultures were incubated in highmoisture air with 5% CO

2at 37°C. The medium

was changed routinely three times a week.

2.2. Preparation of liposomes containing 10Bcompound

A cationic empty liposome (COATSOME EL-C-01: Nichiyu liposome Co.Ltd.) is composed withL-a-dipalmitoyl phosphatidylcholine (26 lmol),cholesterol (20 lmol), and stealylamine (4 lmol).0.5 ml of 100 mM sodium salt of undecahydro-mercaptoclosododecaborate (Na

210B

12H

11SH; called

BSH: Wako Chemical Co. Ltd.) solution to theCOATSOME EL-C-01. The 10B-liposome wasmade by adding solution. The 10B-liposome solu-tion is mixed with BSH solution and 10B-cationicliposome, and is called 10B-liposome solution here-after.

2.3. Mice and tumor injection

Male BALB/c nu/nu mice (Nihon SLC) of sim-ilar age, 6—7 weeks, and similar weight were se-lected. The AsPC-1 (1]107) cells were injected sub-cutaneously into the back of the mice. The mice

H. Yanagie et al./Nuclear Instruments and Methods in Physics Research A 424 (1999) 122—128 123

VI. APPLICATIONS II

were housed in plastic cages and maintained in anair-conditioned room. At 10—15 days after injec-tion, when the estimated tumor weight reached100—300 mg, 0.2 ml of 10B-liposome solution as in-jected intravenously. The mice were sacrificed, 3,6and 12 h after the 10B-liposome solution was injec-ted. The procedures for the tumor implantationand the sacrifice of the animals were in accordancewith approved guidelines of the Institution’s Ani-mal Ethics Committee.

2.4. Preparation of sliced mice samples

The sacrificed mice were frozen at !60°C. Sub-sequently, the frozen mice were cut saggitally into40 lm thick sections and put on the mending tape,freeze-dried at !20°C for 2 weeks, and air driedfor one more week.

2.5. Preparation of standard samples

Boron-containing standard samples were alsoprepared using drying filter paper sheets wetted byBSH solutions of five different 10B concentrationsof relative scale; 1, 10~1, 10~2, 10~3 and 10~4,respectively, where the concentration of 1 corre-sponds to the concentration 1.58]104 ppm of10B compounds. The 10B concentrations of 10B-liposome solution and 10BSH solutions were deter-mined by the prompt c-ray spectometry at theResearch Reactor Institute, Kyoto University [6].

2.6. Neutron irradiations

The whole-body sections are put in close contactwith the CR 39 track etch detector plates (HARZ-LAS; Fukuvi Chemical Industry) using thin adhes-ive tape. The set of mouse samples and standardsamples were simultaneously exposed at two differ-ent irradiation positions according to their purposes.

For visible observation of track image, thermalneutron flux of a neutron exposure of 1012 n/cm2

was required and the samples were attached ontothe surface near the center hole of the thermalcolumn output face (1.2 m]1.2 m) in the TRIGA-II reactor of the Rikkyo University (RUR). Theneutron flux has been measured to be 1]108 n/(cm2 s) and Cd ratio was 6400.

For counting the tracks, the tangential beamport No. 2 with a flux of 1.5]106 n/(cm2 s) andwith the Cd ratio of 2.0 was used. The total fluencewas varied between 4.5]107 and 2.7]1010 n/cm2,depending on the exposure time.

2.7. Etching procedure

Two different etching processes were applied inthis experiment corresponding to different pur-poses. For a-autoradiographic imaging includingproton tracks produced by 14N(n,p) reaction,where 14N is the biogenically abundant nuclide, theCR-39 detector plates were etched in a 7 N NaOHsolution at 70°C for 2 h to reveal tracks. If theneutron component is significantly present in thebeam, the a-autoradiographic image is also con-taminated by the recoil protons from fast neutronscattering on hydrogen in the tissue and the de-tector plate. The tracks were measured by a semi-automatic image analysis system consisting ofa CCD camera, a personal computer and an opticalmicroscope. The NaOH etching method is com-monly used to etch the CR-39 detector. The etchedplates show both a and proton track imageswith slightly diferent contrast. For quantitativeestimation of 10B concentration, however, thebackground tracks induced by protons disturb theanalysis.

A considerable desensitization of track sensitiv-ity of CR-39 can be achieved by etching using PEWsolution [7]. The desensitization of sensitivity ismore effective for low LET particles such as proto-ns. In order to selectively desensitize and eliminateundesirable proton tracks, we applied PEW solu-tion (15 wt% KOH#65 wt% C

2H

5OH#20 wt%

H2O) at 50°C for 8 min to the etching of the CR-39

detector plates.

3. Results and discussion

Recently, positively charged cationic liposomeswere also shown to efficiently introduce genes intocells by forming complexes with the plasmid DNAin solution, facilitating fusion of the liposome/DNAcomplexes with the cell membrane [8]. Yu et al.had reported that they used cationic liposomes to

124 H. Yanagie et al./Nuclear Instruments and Methods in Physics Research A 424 (1999) 122—128

directly deliver the E1A gene into ovarian cancersin mice by intraperitoneal injection of the lipo-some/E1A mixture and found that the treated micesurvived significantly longer than the control micethat received no appropriate treatment [9].

One of the extremely important concerns inBNCT is the 10B delivery system, for which lipo-somes are very attractive and interesting. It is com-monly said that the size of multilamellar liposome(MLV) is about 300 nm, so these MLV liposomesare easily phagocytized by the reticulo-endothelialsystem (RES). So it is very difficult to accumulatethe content of liposome into the target cancers. Toescape these phagocytosis of RES, we have tried toprepare single unilamellar boronated cationic lipo-somes which are used in gene therapy.

3.1. Track etch imaging and desensitization ofproton tracks

Fig. 1 shows whole-body sections of neutroncapture radiograph from a set of AsPC-1 pancre-atic cancer-bearing mice that have been intra-venously injected with 3.2 mg of 10B-liposomesolution. The slices of sacrified and frozen micewere prepared 3 and 12 h after the injection. TheNCAR image of the prepared slice and of the stan-dard samples are shown in this figure, where thesamples were exposed to 1.8]1012 n/cm2 at thethermal column output face and the detector plateswere etched by the NaOH method. It is readilyapparent that the tumor contains high level ofboron until 12 h after injection. There are also areaswithin the liver which contain a high level of boron3 h after injection, however the concentration ofboron decreases with time.

In Fig. 2a track size distributions for CR-39etched by the NaOH method, and (b) by the PEWmethod, are shown. In both cases, the CR-39detector plates attached together with the set ofstandard samples containing 1.58]104 ppm of10B-compounds were exposed to thermal neutronswith a fluence of 4.5]108 n/cm2.

Two peaks of track size appear in the case ofetching in NaOH solution as shown in Fig. 2a. Thelower peak corresponds to proton tracks. The high-er one is due to a- and Li-tracks. It is consideredthat the contribution of Li tracks is not so large

Fig. 1.

because of the short range of Li particles. In orderto discriminate a- and Li-tracks, we will try themultistep etching which would be able to preserveeven for the tracks of shorter range. In the case ofetching using the PEW solution, the greater part ofproton tracks disappeared and only one peak dueto alpha tracks was observed as shown in Fig. 2b.In addition to the desensitization effect, them PEWsolution markedly increases the bulk etching rateand the quality of etched surface of the CR-39 plate.Consequently, the undesirable proton tracks fora-autoradiographic imaging were effectively desen-sitized as shown in Fig. 2b and the 10B biodistribu-tions in whole-body sections of mice were alsoimaged with good quality as in Fig. 1.

The track registration sensitivity of the plastictrack detector depends on restricted energy loss(REL) of incident particles [10]. This means thatthe track sizes of alpha particles are larger than thatof protons. The track-size distributions as shown inFig. 2 were measured using the semi-automatic im-age analysis system. When CR-39 detector platesare etched using the PEW solution, the greater partof proton tracks are desensitized and disappear.The tracks observed are almost all due to alphaparticles originating from 10B(n,a) 7Li reaction, but


VI. APPLICATIONS II

Fig. 2. Track size distributions for CR-39 etched by (a) the NaOH method and (b) the PEW method.

the proton tracks still remain to some extent. Theelimination of the remaining proton backgroundtracks is also possible if we use the discriminationtechnique of the difference of track sizes betweena-tracks and proton tracks.

As shown in Fig. 2b, the proton tracks haveshrunk markedly compared to the alpha tracks,apparently due to the desensitization effect of thePEW etching. Therefore, the discrimination be-tween a tracks and proton tracks has been achieved


Fig. 3.

easily. This enabled us to quantitatively estimatethe 10B concentrations within the tissue sections bycomparison with the standard samples. Fig. 3shows the a-track densities under the calibrationstandard papers as functions of the neutron fluenceand the assumed relative 10B concentrations, wherethe relative 10B concentration of 100 equals1.58]104 ppm of 10B-compound. When sampleswere exposed to higher neutron fluences, the rela-tion between track density and boron concentra-tion lost its linearity due to the increase in thenumber of overlapped tracks. The neutron fluenceof less than 109 n/cm2 was not sufficient to get goodimages of 10B biodistributions in whole-body sec-tions of mice. According to Fig. 3 and the quality ofthe images, it was found that the optimum neutronfluence for both the10B concentration measure-ments and the imaging in the interval between 15and 1500 ppm ranges between 109 and 1010 n/cm2.

3.2. Track density analysis

The samples of mice sacrified at 3 and 6 h afterdirect injections of 1.58]104 ppm of 10B-com-pound solution to the cancer tumor were preparedand exposed to a neutron beam with a fluence of2.7]1010 n/cm2. The CR-39 plate after beingetched in the PEW solution at 50°C for 8 min wasscanned, and the track densities were measured inthe tumor and in the liver. Observed track densities

were measured in the tumor ranged from(7.82$0.24)]103 to (5.48$0.23)]104 tracks/mm2

for the sample sacrified 3 h after the injection of the10B-compound solution and from (6.17$0.59)]103to (3.14$0.68)]104 tracks/mm2 for the samplesacrified 6 h after injection. Average track densityin the image of the liver sacrificed 3 h after injectionwas (4.10$0.13)]103 tracks/mm2, but the imageof the liver sacrified 6 h after injection could not beclearly identified. Therefore, we measured tracksreferring to the position of the liver and the tumorin the full-scale sketch of the section and the posi-tion of the radiographic image of the tumor, thenthe track density of (1.55$0.27)]102 tracks/mm2

was estimated in the portion of the liver.From the observed track densities and Fig. 3, we

can estimate the 10B accumulations in the organs.10B accumulations of 2050 and 83.7 ppm were esti-mated for the strongly and weakly concentratedpart of the tumor at 3 h after injection, respectively.This result promises that with CR-39 radiographyusing track counting it is possible to determine themicro- and fine structure, i.e. micro-autoradiogra-phy, of 10B distribution in the tumor [11].

For instances, accumulation of 41.9 ppm of 10Batoms was estimated in the liver 3 h after injection.For the section taken 6 h after injection, 10B accu-mulations of 695.2 and 66.4 ppm were calculatedfor the strongly and weakly concentrated portion ofthe tumor, respectively and 10B accumulation inthe liver was probably less than 2 ppm.

4. Conclusion

Two types of techniques to describe 10B distribu-tions and area densities were successfully examinedin this study. The NaOH method was applied toobserve visually qualitative distribution of 10B. Thevisible image shows the 10B rich regions at thetumor site and at other organs, especially the liverwith different 10B concentration depending on thetime required after the injection 10B-liposomesolution. The image can also describe organs inthe whole-body section by means of proton trackswith different contrast in comparison to the a-trackimage. The background proton track image is effec-tive to identify the position of the tumor.


VI. APPLICATIONS II

If we want to determine 10B distribution moreexactly, the NaOH method was no more suitablethan the PEW methods because of the interferenceof the proton tracks. Thus, 10B accumulation in thetumor was clearly visible using the neutron captureautoradiography. We found that the 10B cationicliposomes have the possibility of retention to thetumor cells and providing sufficient 10B atoms intothe tumor cells by endocytosis. Transfection effi-ciency of cationic liposome systems can be signifi-cantly improved when complexed with a ligandsuch as transferrin. The presence of the ligand facil-itates the entry of DNA into cells through recep-tor-mediated endocytosis [12].

Maruyama et al. have demonstrated that poly-ethylene glycol binding liposomes of small size(about 100 nm mean diameter) and rigid lipid com-position showed significantly greater accumulationin solid tumor [13]. To escape phagocytosis by theRES, we have tried to prepare boronated polyethy-lene glycol binding BSA and immunoliposomes(stealth immunoliposomes) [14]. Experiments withthese newly 10B delivery systems are in progress forapplication of clinical BNCT trials.

The accurate measurement of 10B distributionsin biological samples with a sensitivity in the ppmrange is essential for evaluating the potential use-fulness of various boron-containing compounds forBNCT. 10B distribution at the cellular level is alsoimportant because the effect on the tumor cells ofheavy particles resulting from 10B (n,a)7Li reactionvaried according to the location of the 10B in the

tumor cells [15,16]. We also continue to study themicrodosimetry of 10B atoms using the “micro-autoradiography” of 10B distribution in tumorcells. We can deliver the 10B atoms to the cytop-lasm and nucleus, selectively, by intelligent drugdelivery system according to the information ofdosimetry by microradiography. Therefore, we willbe able to apply these new techniques for effectiveBNCT for cancer.

References

[1] T. Tomita et al., Biochem. Biophys. Acta 978 (1989) 185.[2] H. Yanagie et al., Br. J. Cancer 63 (1991) 522.[3] H. Yanagie et al., Br. J. Cancer 75 (1997) 660.[4] E.A. Forssen et al., Cancer Res. 52 (1992) 3255.[5] J. Vieweg et al., Cancer Res. 55 (1995) 2366.[6] T. Kobayashi et al., Nucl. Instr and Meth. 204 (1983) 525.[7] G. Somogyi, I. Hunyadi, Proc. 10th Int. Conf. on Solid

State Nuclear Track Detectors, Lyon, 1979, pp. 443-452.[8] D.L. Reimer et al., J. Biol. Chem. 272 (1997) 19480.[9] D. Yu et al., Oncogene 11 (1995) 1383—1388.

[10] E.V. Benton, US Naval Radiological Defence Laboratory,San Francisco, Report USNRDL-TR-67-80.

[11] B. Larsson, Hadrontherapy in Oncology, Elsevier, Amster-dam, 1994, pp. 697—703.

[12] L. Xu, Hum. Gene Ther. 8 (1997) 467.[13] K. Maruyama et al., Proc. 5th Int. Conf. on Applications

of Nuclear Techniques, Washington, USA, SPIE, 2867,1997, pp. 101—104.

[14] H. Yanagie et al., Proc. 5th Int. Conf. on Applications ofNuclear Techniques, Washington, USA, SPIE, 2867 1997,pp. 101—104.

[15] J.A. Coderre, Cancer Res. 47 (1987) 6377.[16] M. Abe, J. Nucl. Med. 27 (1986) 677.


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