- Email: [email protected]
Neutron capture autoradiographic determination of 10B distributions and concentrations in biological samples for boron neutron capture therapy
Nuclear Instruments and Methods in Physics Research A 424 (1999) 122—128
Neutron capture autoradiographic determination of B 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 B atoms in the tumor cells. We prepared a cationic liposome entrapped B compound for the delivery system and examined the delivery capacity of B atoms to pancreatic cancer cell, AsPC-1, in vivo. It is required to achieve an accurate measurement of B distributions 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 B biodistribution in sliced whole-body samples of mice. To selectively desensitize undesirable proton tracks, we applied PEW (KOH#C H OH#H O) 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 size selection method. This enabled us to estimate quantitatively the distributions of B concentrations within the tissue sections 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 nuclear track detector; CR-39; Desensitization of proton tracks
1. Introduction
between B and thermal neutrons. This nuclear reaction is as follows:
The cytotoxic effect of boron neutron capture therapy (BNCT) is caused by a nuclear reaction
B#nPLi#He (a)#2.79 MeV (6.3%) PLi*#He (a)#2.31 MeV (93.7%)
* Corresponding author. Tel.: #81-3-5449-5352; fax: #813-5449-5439; e-mail: [email protected].
PLi#He (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
H. Yanagie et al./Nuclear Instruments and Methods in Physics Research A 424 (1999) 122—128
The resultant Li particles and a particles are high linear energy transfer (LET) particles with relatively high biological efficiency. These particles (a and Li) destroy cells within about 10 lm path length from the site of capture reaction. It is necessary to accumulate B compounds selectively in tumor cells for selective cyototoxicity of tumor cells without affecting adjacent healthy cells. Liposomes have been investigated extensively as carriers for anticancer drugs in attempts to direct active agents to tumors or to protect sensitive tissues form toxicity [1]. Liposomes are useful drug carriers, and its is possible to carry a large amount of B compound in a liposome, which can be delivered to tumor cells. We have reported that B atoms delivered by immunoliposomes are cytotoxic to human pancreatic carcinoma cells (AsPC-1) with thermal neutron irradiation in vitro [2]. The intratumoral injection of boronated immunoliposomes can increase the retention of B atoms in tumor cells, causing tumor growth suppression in vivo under thermal neutron irradiation [3]. Multilamellar vesical immunoliposomes are easily phagocytized by the reticuloendothelial system (RES), so it is very difficult to accumulate the liposomal contents in the target cancers in vivo. Forssen et al. have reported that small unilammelar vesicles consisting of highly purified distearoyl phosphatidylcholine and cholesterol can produce a 10-fold increased delivery of daunorubicin to solid tumors in vivo [4]. Recently, positively charged cationic liposomes were also shown to efficiently introduce genes into cells by forming complexes with the plasmid DNA in solution, facilitating fusion of the liposome/DNA complex with the cell membrane. Cationic liposomes are composed of positively charged lipid bilayers, so they can be bound easily to tumor cells which have acquired a net negative charge [5]. The accurate measurement of B distributions in biological samples with a sensitivity in the ppm range is essential for evaluating the potential usefulness of various boron-containing compounds for BNCT. In this study, we prepared the cationic liposomes as the effective B carrier. To test the validity, we employed a technique of neutron capture autoradiography using track etch detectors
123
(SSNTDs). The B biodistribution was qualitatively and quantitatively determined in whole-body samples of mice. In the alpha-track etch radiographic analysis, alpha tracks were discriminated from proton tracks by carefully selecting the etching solution and the etching condition. The distribution and concentration of B within the tissue sections were measured by a microscopic analyzing system comparing with the results using standard samples.
2. Materials and methods 2.1. Preparation of target tumor cells A human pancreatic carcinoma cell line (AsPC-1: Dainihon Seiyaku Co.Ltd.) produces carcinoembryonic antigen (CEA) as follows: The AsPC-1 was maintained in a medium (PRMI 1640: Hazeleton Biologics Inc.) supplemented with 10% fetal calf serum (Cell Culture Labs.) and 100 lg ml\ kanamycin. All cultures were incubated in high moisture air with 5% CO at 37°C. The medium was changed routinely three times a week. 2.2. Preparation of liposomes containing B compound A cationic empty liposome (COATSOME ELC-01: Nichiyu liposome Co.Ltd.) is composed with L-a-dipalmitoyl phosphatidylcholine (26 lmol), cholesterol (20 lmol), and stealylamine (4 lmol). 0.5 ml of 100 mM sodium salt of undecahydromercaptoclosododecaborate (Na B H SH; called BSH: Wako Chemical Co. Ltd.) solution to the COATSOME EL-C-01. The B-liposome was made by adding solution. The B-liposome solution is mixed with BSH solution and B-cationic liposome, and is called B-liposome solution hereafter. 2.3. Mice and tumor injection Male BALB/c nu/nu mice (Nihon SLC) of similar age, 6—7 weeks, and similar weight were selected. The AsPC-1 (1;10) cells were injected subcutaneously into the back of the mice. The mice
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were housed in plastic cages and maintained in an air-conditioned room. At 10—15 days after injection, when the estimated tumor weight reached 100—300 mg, 0.2 ml of B-liposome solution as injected intravenously. The mice were sacrificed, 3,6 and 12 h after the B-liposome solution was injected. The procedures for the tumor implantation and the sacrifice of the animals were in accordance with approved guidelines of the Institution’s Animal Ethics Committee. 2.4. Preparation of sliced mice samples The sacrificed mice were frozen at !60°C. Subsequently, the frozen mice were cut saggitally into 40 lm thick sections and put on the mending tape, freeze-dried at !20°C for 2 weeks, and air dried for one more week. 2.5. Preparation of standard samples Boron-containing standard samples were also prepared using drying filter paper sheets wetted by BSH solutions of five different B concentrations of relative scale; 1, 10\, 10\, 10\ and 10\, respectively, where the concentration of 1 corresponds to the concentration 1.58;10 ppm of B compounds. The B concentrations of Bliposome solution and BSH solutions were determined by the prompt c-ray spectometry at the Research Reactor Institute, Kyoto University [6]. 2.6. Neutron irradiations The whole-body sections are put in close contact with the CR 39 track etch detector plates (HARZLAS; Fukuvi Chemical Industry) using thin adhesive tape. The set of mouse samples and standard samples were simultaneously exposed at two different irradiation positions according to their purposes. For visible observation of track image, thermal neutron flux of a neutron exposure of 10 n/cm was required and the samples were attached onto the surface near the center hole of the thermal column output face (1.2 m;1.2 m) in the TRIGAII reactor of the Rikkyo University (RUR). The neutron flux has been measured to be 1;10 n/ (cm s) and Cd ratio was 6400.
For counting the tracks, the tangential beam port No. 2 with a flux of 1.5;10 n/(cm s) and with the Cd ratio of 2.0 was used. The total fluence was varied between 4.5;10 and 2.7;10 n/cm, depending on the exposure time. 2.7. Etching procedure Two different etching processes were applied in this experiment corresponding to different purposes. For a-autoradiographic imaging including proton tracks produced by N(n,p) reaction, where N is the biogenically abundant nuclide, the CR-39 detector plates were etched in a 7 N NaOH solution at 70°C for 2 h to reveal tracks. If the neutron component is significantly present in the beam, the a-autoradiographic image is also contaminated by the recoil protons from fast neutron scattering on hydrogen in the tissue and the detector plate. The tracks were measured by a semiautomatic image analysis system consisting of a CCD camera, a personal computer and an optical microscope. The NaOH etching method is commonly used to etch the CR-39 detector. The etched plates show both a and proton track images with slightly diferent contrast. For quantitative estimation of B concentration, however, the background tracks induced by protons disturb the analysis. A considerable desensitization of track sensitivity of CR-39 can be achieved by etching using PEW solution [7]. The desensitization of sensitivity is more effective for low LET particles such as protons. In order to selectively desensitize and eliminate undesirable proton tracks, we applied PEW solution (15 wt% KOH#65 wt% C H OH#20 wt% H O) at 50°C for 8 min to the etching of the CR-39 detector plates.
3. Results and discussion Recently, positively charged cationic liposomes were also shown to efficiently introduce genes into cells by forming complexes with the plasmid DNA in solution, facilitating fusion of the liposome/DNA complexes with the cell membrane [8]. Yu et al. had reported that they used cationic liposomes to
H. Yanagie et al./Nuclear Instruments and Methods in Physics Research A 424 (1999) 122—128
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directly deliver the E1A gene into ovarian cancers in mice by intraperitoneal injection of the liposome/E1A mixture and found that the treated mice survived significantly longer than the control mice that received no appropriate treatment [9]. One of the extremely important concerns in BNCT is the B delivery system, for which liposomes are very attractive and interesting. It is commonly said that the size of multilamellar liposome (MLV) is about 300 nm, so these MLV liposomes are easily phagocytized by the reticulo-endothelial system (RES). So it is very difficult to accumulate the content of liposome into the target cancers. To escape these phagocytosis of RES, we have tried to prepare single unilamellar boronated cationic liposomes which are used in gene therapy. 3.1. Track etch imaging and desensitization of proton tracks Fig. 1 shows whole-body sections of neutron capture radiograph from a set of AsPC-1 pancreatic cancer-bearing mice that have been intravenously injected with 3.2 mg of B-liposome solution. The slices of sacrified and frozen mice were prepared 3 and 12 h after the injection. The NCAR image of the prepared slice and of the standard samples are shown in this figure, where the samples were exposed to 1.8;10 n/cm at the thermal column output face and the detector plates were etched by the NaOH method. It is readily apparent that the tumor contains high level of boron until 12 h after injection. There are also areas within the liver which contain a high level of boron 3 h after injection, however the concentration of boron decreases with time. In Fig. 2a track size distributions for CR-39 etched by the NaOH method, and (b) by the PEW method, are shown. In both cases, the CR-39 detector plates attached together with the set of standard samples containing 1.58;10 ppm of B-compounds were exposed to thermal neutrons with a fluence of 4.5;10 n/cm. Two peaks of track size appear in the case of etching in NaOH solution as shown in Fig. 2a. The lower peak corresponds to proton tracks. The higher one is due to a- and Li-tracks. It is considered that the contribution of Li tracks is not so large
Fig. 1.
because of the short range of Li particles. In order to discriminate a- and Li-tracks, we will try the multistep etching which would be able to preserve even for the tracks of shorter range. In the case of etching using the PEW solution, the greater part of proton tracks disappeared and only one peak due to alpha tracks was observed as shown in Fig. 2b. In addition to the desensitization effect, them PEW solution markedly increases the bulk etching rate and the quality of etched surface of the CR-39 plate. Consequently, the undesirable proton tracks for a-autoradiographic imaging were effectively desensitized as shown in Fig. 2b and the B biodistributions in whole-body sections of mice were also imaged with good quality as in Fig. 1. The track registration sensitivity of the plastic track detector depends on restricted energy loss (REL) of incident particles [10]. This means that the track sizes of alpha particles are larger than that of protons. The track-size distributions as shown in Fig. 2 were measured using the semi-automatic image analysis system. When CR-39 detector plates are etched using the PEW solution, the greater part of proton tracks are desensitized and disappear. The tracks observed are almost all due to alpha particles originating from B(n,a) Li reaction, but
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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. The elimination of the remaining proton background tracks is also possible if we use the discrimination technique of the difference of track sizes between a-tracks and proton tracks.
As shown in Fig. 2b, the proton tracks have shrunk markedly compared to the alpha tracks, apparently due to the desensitization effect of the PEW etching. Therefore, the discrimination between a tracks and proton tracks has been achieved
H. Yanagie et al./Nuclear Instruments and Methods in Physics Research A 424 (1999) 122—128
Fig. 3.
easily. This enabled us to quantitatively estimate the B concentrations within the tissue sections by comparison with the standard samples. Fig. 3 shows the a-track densities under the calibration standard papers as functions of the neutron fluence and the assumed relative B concentrations, where the relative B concentration of 10 equals 1.58;10 ppm of B-compound. When samples were exposed to higher neutron fluences, the relation between track density and boron concentration lost its linearity due to the increase in the number of overlapped tracks. The neutron fluence of less than 10 n/cm was not sufficient to get good images of B biodistributions in whole-body sections of mice. According to Fig. 3 and the quality of the images, it was found that the optimum neutron fluence for both theB concentration measurements and the imaging in the interval between 15 and 1500 ppm ranges between 10 and 10 n/cm. 3.2. Track density analysis The samples of mice sacrified at 3 and 6 h after direct injections of 1.58;10 ppm of B-compound solution to the cancer tumor were prepared and exposed to a neutron beam with a fluence of 2.7;10 n/cm. The CR-39 plate after being etched in the PEW solution at 50°C for 8 min was scanned, and the track densities were measured in the tumor and in the liver. Observed track densities
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were measured in the tumor ranged from (7.82$0.24);10 to (5.48$0.23);10 tracks/mm for the sample sacrified 3 h after the injection of the B-compound solution and from (6.17$0.59);10 to (3.14$0.68);10 tracks/mm for the sample sacrified 6 h after injection. Average track density in the image of the liver sacrificed 3 h after injection was (4.10$0.13);10 tracks/mm, but the image of the liver sacrified 6 h after injection could not be clearly identified. Therefore, we measured tracks referring to the position of the liver and the tumor in the full-scale sketch of the section and the position of the radiographic image of the tumor, then the track density of (1.55$0.27);10 tracks/mm was estimated in the portion of the liver. From the observed track densities and Fig. 3, we can estimate the B accumulations in the organs. B accumulations of 2050 and 83.7 ppm were estimated for the strongly and weakly concentrated part of the tumor at 3 h after injection, respectively. This result promises that with CR-39 radiography using track counting it is possible to determine the micro- and fine structure, i.e. micro-autoradiography, of B distribution in the tumor [11]. For instances, accumulation of 41.9 ppm of B atoms was estimated in the liver 3 h after injection. For the section taken 6 h after injection, B accumulations of 695.2 and 66.4 ppm were calculated for the strongly and weakly concentrated portion of the tumor, respectively and B accumulation in the liver was probably less than 2 ppm.
4. Conclusion Two types of techniques to describe B distributions and area densities were successfully examined in this study. The NaOH method was applied to observe visually qualitative distribution of B. The visible image shows the B rich regions at the tumor site and at other organs, especially the liver with different B concentration depending on the time required after the injection B-liposome solution. The image can also describe organs in the whole-body section by means of proton tracks with different contrast in comparison to the a-track image. The background proton track image is effective to identify the position of the tumor.
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If we want to determine B distribution more exactly, the NaOH method was no more suitable than the PEW methods because of the interference of the proton tracks. Thus, B accumulation in the tumor was clearly visible using the neutron capture autoradiography. We found that the B cationic liposomes have the possibility of retention to the tumor cells and providing sufficient B atoms into the tumor cells by endocytosis. Transfection efficiency of cationic liposome systems can be significantly improved when complexed with a ligand such as transferrin. The presence of the ligand facilitates the entry of DNA into cells through receptor-mediated endocytosis [12]. Maruyama et al. have demonstrated that polyethylene glycol binding liposomes of small size (about 100 nm mean diameter) and rigid lipid composition showed significantly greater accumulation in solid tumor [13]. To escape phagocytosis by the RES, we have tried to prepare boronated polyethylene glycol binding BSA and immunoliposomes (stealth immunoliposomes) [14]. Experiments with these newly B delivery systems are in progress for application of clinical BNCT trials. The accurate measurement of B distributions in biological samples with a sensitivity in the ppm range is essential for evaluating the potential usefulness of various boron-containing compounds for BNCT. B distribution at the cellular level is also important because the effect on the tumor cells of heavy particles resulting from B (n,a)Li reaction varied according to the location of the B in the
tumor cells [15,16]. We also continue to study the microdosimetry of B atoms using the “microautoradiography” of B distribution in tumor cells. We can deliver the B atoms to the cytoplasm and nucleus, selectively, by intelligent drug delivery system according to the information of dosimetry by microradiography. Therefore, we will be able to apply these new techniques for effective BNCT for cancer. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
[14]
[15] [16]
T. Tomita et al., Biochem. Biophys. Acta 978 (1989) 185. H. Yanagie et al., Br. J. Cancer 63 (1991) 522. H. Yanagie et al., Br. J. Cancer 75 (1997) 660. E.A. Forssen et al., Cancer Res. 52 (1992) 3255. J. Vieweg et al., Cancer Res. 55 (1995) 2366. T. Kobayashi et al., Nucl. Instr and Meth. 204 (1983) 525. G. Somogyi, I. Hunyadi, Proc. 10th Int. Conf. on Solid State Nuclear Track Detectors, Lyon, 1979, pp. 443-452. D.L. Reimer et al., J. Biol. Chem. 272 (1997) 19480. D. Yu et al., Oncogene 11 (1995) 1383—1388. E.V. Benton, US Naval Radiological Defence Laboratory, San Francisco, Report USNRDL-TR-67-80. B. Larsson, Hadrontherapy in Oncology, Elsevier, Amsterdam, 1994, pp. 697—703. L. Xu, Hum. Gene Ther. 8 (1997) 467. K. Maruyama et al., Proc. 5th Int. Conf. on Applications of Nuclear Techniques, Washington, USA, SPIE, 2867, 1997, pp. 101—104. H. Yanagie et al., Proc. 5th Int. Conf. on Applications of Nuclear Techniques, Washington, USA, SPIE, 2867 1997, pp. 101—104. J.A. Coderre, Cancer Res. 47 (1987) 6377. M. Abe, J. Nucl. Med. 27 (1986) 677.
Neutron capture autoradiographic determination of B 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 B atoms in the tumor cells. We prepared a cationic liposome entrapped B compound for the delivery system and examined the delivery capacity of B atoms to pancreatic cancer cell, AsPC-1, in vivo. It is required to achieve an accurate measurement of B distributions 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 B biodistribution in sliced whole-body samples of mice. To selectively desensitize undesirable proton tracks, we applied PEW (KOH#C H OH#H O) 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 size selection method. This enabled us to estimate quantitatively the distributions of B concentrations within the tissue sections 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 nuclear track detector; CR-39; Desensitization of proton tracks
1. Introduction
between B and thermal neutrons. This nuclear reaction is as follows:
The cytotoxic effect of boron neutron capture therapy (BNCT) is caused by a nuclear reaction
B#nPLi#He (a)#2.79 MeV (6.3%) PLi*#He (a)#2.31 MeV (93.7%)
* Corresponding author. Tel.: #81-3-5449-5352; fax: #813-5449-5439; e-mail: [email protected].
PLi#He (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
H. Yanagie et al./Nuclear Instruments and Methods in Physics Research A 424 (1999) 122—128
The resultant Li particles and a particles are high linear energy transfer (LET) particles with relatively high biological efficiency. These particles (a and Li) destroy cells within about 10 lm path length from the site of capture reaction. It is necessary to accumulate B compounds selectively in tumor cells for selective cyototoxicity of tumor cells without affecting adjacent healthy cells. Liposomes have been investigated extensively as carriers for anticancer drugs in attempts to direct active agents to tumors or to protect sensitive tissues form toxicity [1]. Liposomes are useful drug carriers, and its is possible to carry a large amount of B compound in a liposome, which can be delivered to tumor cells. We have reported that B atoms delivered by immunoliposomes are cytotoxic to human pancreatic carcinoma cells (AsPC-1) with thermal neutron irradiation in vitro [2]. The intratumoral injection of boronated immunoliposomes can increase the retention of B atoms in tumor cells, causing tumor growth suppression in vivo under thermal neutron irradiation [3]. Multilamellar vesical immunoliposomes are easily phagocytized by the reticuloendothelial system (RES), so it is very difficult to accumulate the liposomal contents in the target cancers in vivo. Forssen et al. have reported that small unilammelar vesicles consisting of highly purified distearoyl phosphatidylcholine and cholesterol can produce a 10-fold increased delivery of daunorubicin to solid tumors in vivo [4]. Recently, positively charged cationic liposomes were also shown to efficiently introduce genes into cells by forming complexes with the plasmid DNA in solution, facilitating fusion of the liposome/DNA complex with the cell membrane. Cationic liposomes are composed of positively charged lipid bilayers, so they can be bound easily to tumor cells which have acquired a net negative charge [5]. The accurate measurement of B distributions in biological samples with a sensitivity in the ppm range is essential for evaluating the potential usefulness of various boron-containing compounds for BNCT. In this study, we prepared the cationic liposomes as the effective B carrier. To test the validity, we employed a technique of neutron capture autoradiography using track etch detectors
123
(SSNTDs). The B biodistribution was qualitatively and quantitatively determined in whole-body samples of mice. In the alpha-track etch radiographic analysis, alpha tracks were discriminated from proton tracks by carefully selecting the etching solution and the etching condition. The distribution and concentration of B within the tissue sections were measured by a microscopic analyzing system comparing with the results using standard samples.
2. Materials and methods 2.1. Preparation of target tumor cells A human pancreatic carcinoma cell line (AsPC-1: Dainihon Seiyaku Co.Ltd.) produces carcinoembryonic antigen (CEA) as follows: The AsPC-1 was maintained in a medium (PRMI 1640: Hazeleton Biologics Inc.) supplemented with 10% fetal calf serum (Cell Culture Labs.) and 100 lg ml\ kanamycin. All cultures were incubated in high moisture air with 5% CO at 37°C. The medium was changed routinely three times a week. 2.2. Preparation of liposomes containing B compound A cationic empty liposome (COATSOME ELC-01: Nichiyu liposome Co.Ltd.) is composed with L-a-dipalmitoyl phosphatidylcholine (26 lmol), cholesterol (20 lmol), and stealylamine (4 lmol). 0.5 ml of 100 mM sodium salt of undecahydromercaptoclosododecaborate (Na B H SH; called BSH: Wako Chemical Co. Ltd.) solution to the COATSOME EL-C-01. The B-liposome was made by adding solution. The B-liposome solution is mixed with BSH solution and B-cationic liposome, and is called B-liposome solution hereafter. 2.3. Mice and tumor injection Male BALB/c nu/nu mice (Nihon SLC) of similar age, 6—7 weeks, and similar weight were selected. The AsPC-1 (1;10) cells were injected subcutaneously into the back of the mice. The mice
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were housed in plastic cages and maintained in an air-conditioned room. At 10—15 days after injection, when the estimated tumor weight reached 100—300 mg, 0.2 ml of B-liposome solution as injected intravenously. The mice were sacrificed, 3,6 and 12 h after the B-liposome solution was injected. The procedures for the tumor implantation and the sacrifice of the animals were in accordance with approved guidelines of the Institution’s Animal Ethics Committee. 2.4. Preparation of sliced mice samples The sacrificed mice were frozen at !60°C. Subsequently, the frozen mice were cut saggitally into 40 lm thick sections and put on the mending tape, freeze-dried at !20°C for 2 weeks, and air dried for one more week. 2.5. Preparation of standard samples Boron-containing standard samples were also prepared using drying filter paper sheets wetted by BSH solutions of five different B concentrations of relative scale; 1, 10\, 10\, 10\ and 10\, respectively, where the concentration of 1 corresponds to the concentration 1.58;10 ppm of B compounds. The B concentrations of Bliposome solution and BSH solutions were determined by the prompt c-ray spectometry at the Research Reactor Institute, Kyoto University [6]. 2.6. Neutron irradiations The whole-body sections are put in close contact with the CR 39 track etch detector plates (HARZLAS; Fukuvi Chemical Industry) using thin adhesive tape. The set of mouse samples and standard samples were simultaneously exposed at two different irradiation positions according to their purposes. For visible observation of track image, thermal neutron flux of a neutron exposure of 10 n/cm was required and the samples were attached onto the surface near the center hole of the thermal column output face (1.2 m;1.2 m) in the TRIGAII reactor of the Rikkyo University (RUR). The neutron flux has been measured to be 1;10 n/ (cm s) and Cd ratio was 6400.
For counting the tracks, the tangential beam port No. 2 with a flux of 1.5;10 n/(cm s) and with the Cd ratio of 2.0 was used. The total fluence was varied between 4.5;10 and 2.7;10 n/cm, depending on the exposure time. 2.7. Etching procedure Two different etching processes were applied in this experiment corresponding to different purposes. For a-autoradiographic imaging including proton tracks produced by N(n,p) reaction, where N is the biogenically abundant nuclide, the CR-39 detector plates were etched in a 7 N NaOH solution at 70°C for 2 h to reveal tracks. If the neutron component is significantly present in the beam, the a-autoradiographic image is also contaminated by the recoil protons from fast neutron scattering on hydrogen in the tissue and the detector plate. The tracks were measured by a semiautomatic image analysis system consisting of a CCD camera, a personal computer and an optical microscope. The NaOH etching method is commonly used to etch the CR-39 detector. The etched plates show both a and proton track images with slightly diferent contrast. For quantitative estimation of B concentration, however, the background tracks induced by protons disturb the analysis. A considerable desensitization of track sensitivity of CR-39 can be achieved by etching using PEW solution [7]. The desensitization of sensitivity is more effective for low LET particles such as protons. In order to selectively desensitize and eliminate undesirable proton tracks, we applied PEW solution (15 wt% KOH#65 wt% C H OH#20 wt% H O) at 50°C for 8 min to the etching of the CR-39 detector plates.
3. Results and discussion Recently, positively charged cationic liposomes were also shown to efficiently introduce genes into cells by forming complexes with the plasmid DNA in solution, facilitating fusion of the liposome/DNA complexes with the cell membrane [8]. Yu et al. had reported that they used cationic liposomes to
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directly deliver the E1A gene into ovarian cancers in mice by intraperitoneal injection of the liposome/E1A mixture and found that the treated mice survived significantly longer than the control mice that received no appropriate treatment [9]. One of the extremely important concerns in BNCT is the B delivery system, for which liposomes are very attractive and interesting. It is commonly said that the size of multilamellar liposome (MLV) is about 300 nm, so these MLV liposomes are easily phagocytized by the reticulo-endothelial system (RES). So it is very difficult to accumulate the content of liposome into the target cancers. To escape these phagocytosis of RES, we have tried to prepare single unilamellar boronated cationic liposomes which are used in gene therapy. 3.1. Track etch imaging and desensitization of proton tracks Fig. 1 shows whole-body sections of neutron capture radiograph from a set of AsPC-1 pancreatic cancer-bearing mice that have been intravenously injected with 3.2 mg of B-liposome solution. The slices of sacrified and frozen mice were prepared 3 and 12 h after the injection. The NCAR image of the prepared slice and of the standard samples are shown in this figure, where the samples were exposed to 1.8;10 n/cm at the thermal column output face and the detector plates were etched by the NaOH method. It is readily apparent that the tumor contains high level of boron until 12 h after injection. There are also areas within the liver which contain a high level of boron 3 h after injection, however the concentration of boron decreases with time. In Fig. 2a track size distributions for CR-39 etched by the NaOH method, and (b) by the PEW method, are shown. In both cases, the CR-39 detector plates attached together with the set of standard samples containing 1.58;10 ppm of B-compounds were exposed to thermal neutrons with a fluence of 4.5;10 n/cm. Two peaks of track size appear in the case of etching in NaOH solution as shown in Fig. 2a. The lower peak corresponds to proton tracks. The higher one is due to a- and Li-tracks. It is considered that the contribution of Li tracks is not so large
Fig. 1.
because of the short range of Li particles. In order to discriminate a- and Li-tracks, we will try the multistep etching which would be able to preserve even for the tracks of shorter range. In the case of etching using the PEW solution, the greater part of proton tracks disappeared and only one peak due to alpha tracks was observed as shown in Fig. 2b. In addition to the desensitization effect, them PEW solution markedly increases the bulk etching rate and the quality of etched surface of the CR-39 plate. Consequently, the undesirable proton tracks for a-autoradiographic imaging were effectively desensitized as shown in Fig. 2b and the B biodistributions in whole-body sections of mice were also imaged with good quality as in Fig. 1. The track registration sensitivity of the plastic track detector depends on restricted energy loss (REL) of incident particles [10]. This means that the track sizes of alpha particles are larger than that of protons. The track-size distributions as shown in Fig. 2 were measured using the semi-automatic image analysis system. When CR-39 detector plates are etched using the PEW solution, the greater part of proton tracks are desensitized and disappear. The tracks observed are almost all due to alpha particles originating from B(n,a) Li reaction, but
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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. The elimination of the remaining proton background tracks is also possible if we use the discrimination technique of the difference of track sizes between a-tracks and proton tracks.
As shown in Fig. 2b, the proton tracks have shrunk markedly compared to the alpha tracks, apparently due to the desensitization effect of the PEW etching. Therefore, the discrimination between a tracks and proton tracks has been achieved
H. Yanagie et al./Nuclear Instruments and Methods in Physics Research A 424 (1999) 122—128
Fig. 3.
easily. This enabled us to quantitatively estimate the B concentrations within the tissue sections by comparison with the standard samples. Fig. 3 shows the a-track densities under the calibration standard papers as functions of the neutron fluence and the assumed relative B concentrations, where the relative B concentration of 10 equals 1.58;10 ppm of B-compound. When samples were exposed to higher neutron fluences, the relation between track density and boron concentration lost its linearity due to the increase in the number of overlapped tracks. The neutron fluence of less than 10 n/cm was not sufficient to get good images of B biodistributions in whole-body sections of mice. According to Fig. 3 and the quality of the images, it was found that the optimum neutron fluence for both theB concentration measurements and the imaging in the interval between 15 and 1500 ppm ranges between 10 and 10 n/cm. 3.2. Track density analysis The samples of mice sacrified at 3 and 6 h after direct injections of 1.58;10 ppm of B-compound solution to the cancer tumor were prepared and exposed to a neutron beam with a fluence of 2.7;10 n/cm. The CR-39 plate after being etched in the PEW solution at 50°C for 8 min was scanned, and the track densities were measured in the tumor and in the liver. Observed track densities
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were measured in the tumor ranged from (7.82$0.24);10 to (5.48$0.23);10 tracks/mm for the sample sacrified 3 h after the injection of the B-compound solution and from (6.17$0.59);10 to (3.14$0.68);10 tracks/mm for the sample sacrified 6 h after injection. Average track density in the image of the liver sacrificed 3 h after injection was (4.10$0.13);10 tracks/mm, but the image of the liver sacrified 6 h after injection could not be clearly identified. Therefore, we measured tracks referring to the position of the liver and the tumor in the full-scale sketch of the section and the position of the radiographic image of the tumor, then the track density of (1.55$0.27);10 tracks/mm was estimated in the portion of the liver. From the observed track densities and Fig. 3, we can estimate the B accumulations in the organs. B accumulations of 2050 and 83.7 ppm were estimated for the strongly and weakly concentrated part of the tumor at 3 h after injection, respectively. This result promises that with CR-39 radiography using track counting it is possible to determine the micro- and fine structure, i.e. micro-autoradiography, of B distribution in the tumor [11]. For instances, accumulation of 41.9 ppm of B atoms was estimated in the liver 3 h after injection. For the section taken 6 h after injection, B accumulations of 695.2 and 66.4 ppm were calculated for the strongly and weakly concentrated portion of the tumor, respectively and B accumulation in the liver was probably less than 2 ppm.
4. Conclusion Two types of techniques to describe B distributions and area densities were successfully examined in this study. The NaOH method was applied to observe visually qualitative distribution of B. The visible image shows the B rich regions at the tumor site and at other organs, especially the liver with different B concentration depending on the time required after the injection B-liposome solution. The image can also describe organs in the whole-body section by means of proton tracks with different contrast in comparison to the a-track image. The background proton track image is effective to identify the position of the tumor.
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If we want to determine B distribution more exactly, the NaOH method was no more suitable than the PEW methods because of the interference of the proton tracks. Thus, B accumulation in the tumor was clearly visible using the neutron capture autoradiography. We found that the B cationic liposomes have the possibility of retention to the tumor cells and providing sufficient B atoms into the tumor cells by endocytosis. Transfection efficiency of cationic liposome systems can be significantly improved when complexed with a ligand such as transferrin. The presence of the ligand facilitates the entry of DNA into cells through receptor-mediated endocytosis [12]. Maruyama et al. have demonstrated that polyethylene glycol binding liposomes of small size (about 100 nm mean diameter) and rigid lipid composition showed significantly greater accumulation in solid tumor [13]. To escape phagocytosis by the RES, we have tried to prepare boronated polyethylene glycol binding BSA and immunoliposomes (stealth immunoliposomes) [14]. Experiments with these newly B delivery systems are in progress for application of clinical BNCT trials. The accurate measurement of B distributions in biological samples with a sensitivity in the ppm range is essential for evaluating the potential usefulness of various boron-containing compounds for BNCT. B distribution at the cellular level is also important because the effect on the tumor cells of heavy particles resulting from B (n,a)Li reaction varied according to the location of the B in the
tumor cells [15,16]. We also continue to study the microdosimetry of B atoms using the “microautoradiography” of B distribution in tumor cells. We can deliver the B atoms to the cytoplasm and nucleus, selectively, by intelligent drug delivery system according to the information of dosimetry by microradiography. Therefore, we will be able to apply these new techniques for effective BNCT for cancer. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
[14]
[15] [16]
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