Skip to main content
NCBI home page
Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2015 Dec 9;142(4):767–775. doi: 10.1007/s00432-015-2085-0

In vivo evaluation of neutron capture therapy effectivity using calcium phosphate-based nanoparticles as Gd-DTPA delivery agent

Novriana Dewi 1, Peng Mi 2,3,4, Hironobu Yanagie 1,5,6,✉, Yuriko Sakurai 5, Yasuyuki Morishita 7, Masashi Yanagawa 8, Takayuki Nakagawa 9, Atsuko Shinohara 10, Takehisa Matsukawa 11, Kazuhito Yokoyama 11, Horacio Cabral 12, Minoru Suzuki 13, Yoshinori Sakurai 13, Hiroki Tanaka 13, Koji Ono 13, Nobuhiro Nishiyama 2,3, Kazunori Kataoka 2,4,14, Hiroyuki Takahashi 1,5
PMCID: PMC11819321  PMID: 26650198

Abstract

Purpose

A more immediate impact for therapeutic approaches of current clinical research efforts is of major interest, which might be obtained by developing a noninvasive radiation dose-escalation strategy, and neutron capture therapy represents one such novel approach. Furthermore, some recent researches on neutron capture therapy have focused on using gadolinium as an alternative or complementary for currently used boron, taking into account several advantages that gadolinium offers. Therefore, in this study, we carried out feasibility evaluation for both single and multiple injections of gadolinium-based MRI contrast agent incorporated in calcium phosphate nanoparticles as neutron capture therapy agent.

Methods

In vivo evaluation was performed on colon carcinoma Col-26 tumor-bearing mice irradiated at nuclear reactor facility of Kyoto University Research Reactor Institute with average neutron fluence of 1.8 × 1012 n/cm2. Antitumor effectivity was evaluated based on tumor growth suppression assessed until 27 days after neutron irradiation, followed by histopathological analysis on tumor slice.

Results

The experimental results showed that the tumor growth of irradiated mice injected beforehand with Gd-DTPA-incorporating calcium phosphate-based nanoparticles was suppressed up to four times higher compared to the non-treated group, supported by the results of histopathological analysis.

Conclusion

The results of antitumor effectivity observed on tumor-bearing mice after neutron irradiation indicated possible effectivity of gadolinium-based neutron capture therapy treatment.

Keywords: Neutron capture therapy, Gadolinium, Nanoparticles, Calcium phosphate

Introduction

Neutron capture therapy (NCT) is a radiation therapy utilizing secondary radiation particles produced after neutron capture reaction in killing the cancer cells. In NCT, neutron beam is applied to the region of interest where the target tissue contains a relatively high concentration of neutron absorber compounds. Reaction between these compounds with neutron creates secondary products that deposit most of their dose locally, sparing the surrounding normal tissues. Neutron capture therapy was introduced soon after the discovery of neutron (Locher 1936) and has been through continuous advancement in different fields of study. Currently, 10B-based compounds are being used for clinical trial in several NCT facilities in the world and have been showing promising results on patient with difficult-to-treat cancer type (Henriksson et al. 2008; Yamamoto et al. 2004; Chadha et al. 1998; Fuwa et al. 2008; Joensuu et al. 2003).

Boron-10 isotope undergoes (n,α) reaction after neutron capture, which then splits into energetic alpha particle and lithium nucleus. These secondary particles have combined path length of approximately 12 microns, which is about the same size of one cell diameter. However, the short range of these secondary particles may not reach the nuclei of some nearby cells, which might increase the possibility of cancer recurrent. Therefore, homogenous delivery of 10B atom to every tumor cell nucleus is necessary to obtain the desired therapeutic effect of boron neutron capture therapy (BNCT), which might be difficult to achieve.

The use of gadolinium as an alternative of NCT agent has been getting attention due to the high neutron cross section of 157Gd isotope (255,000 barns) as the highest thermal neutron cross section among all stable nuclides, around 66 times larger compared to that of boron. This property makes it possible to decrease the total neutron fluence needed for the same number of thermal neutron absorptions with 10B. However, this requires the products of gadolinium neutron capture reaction (GdNCR) to be as damaging as the alpha particle and lithium ions from boron neutron capture reaction (BNCR) to produce equivalent biological effect in the target (Goorley and Nikjoo 2000).

Gadolinium neutron capture reaction releases a complex spectrum of secondary particles, including long-range gammas, low-energy internal conversion electrons, Auger electrons, and characteristics X-rays. The photons emitted in the (n,γ) reactions deposit energy over a longer path length than the products from BNCR and is regarded as limiting the localization of therapeutic effectiveness. However, if 157Gd uptake is limited to tumor volume in the order of some cm3, then an additional effect might be added (Cerullo et al. 2009). These properties of GdNCR can also increase the possibility of killing tumor cells even when gadolinium is accumulated outside the tumor cells, thus eliminating the requirement of intracellular delivery of gadolinium (De Stasio et al. 2001). It has been previously reported by Hambley and Hait (2009) that drugs generally do not penetrate further than three to five cell diameters from blood vessels, which leaves distant tumor cells with low concentration or even without any drugs. Thus, the drawback of longer-range gamma rays produced after neutron capture reaction by gadolinium could be optimized to increase the chance of hitting all the cells in the tumor.

Gadolinium-based neutron capture therapy (GdNCT) was first formulated in the 1980s (Brugger and Shih 1989; Martin et al. 1989). However, its development has suffered due to the lack of appropriate Gd-containing tumor-selective agents (Masiakowski et al. 1992). Its clinical application has been rather limited because of the difficulty in retaining a sufficient amount of gadolinium in tumors during the neutron irradiation (Shih and Brugger 1992). Gadolinium ion (Gd3+) is also known to be toxic and must be chemically stabilized by chelation. Therefore, standard gadolinium-containing MRI contrast agents should be considered as the most realistic option for GdNCT agent, since their pharmacology has been extensively studied and has already been approved for clinical use. Nevertheless, most of gadolinium-based MRI contrast agents are reported to have short blood circulation time and low specificity in tissue accumulation (Weinmann et al. 1984; Aime and Caravan 2009). Therefore, an efficient drug delivery system is indispensable to accumulate and maintain sufficient amount of gadolinium into tumor site during neutron irradiation. It also should be capable of establishing high tumor-to-normal tissue (T/N) and tumor-to-blood (T/B) concentration ratio in order to deliver high radiation dose to the tumor with tolerable dose to healthy tissue.

Particulate carriers such as emulsion, liposome, and nanoparticles have been of interest for drug delivery system considering that they posses the characteristics necessary to enhance drug localization as well as drug release in tumor site. Our group first applied liposomes in BNCT and had shown that immune-liposome could serve as selective and efficient carriers of 10B atoms to target tumor cells (Yanagie et al. 1991), followed by continuous work on drug delivery system for BNCT (Yanagie et al. 2008). Our previous results of tumor growth suppression with gadolinium-encapsulating liposome (Dewi et al. 2013) as well as some other reports on nanoparticles encapsulating gadolinium-based compound (Tokumitsu et al. 2000; Watanabe et al. 2002; Ichikawa et al. 2014) have also shown promising feasibility of MRI contrast agents for GdNCT.

Nanoparticles are getting attention as drug carrier device because of its capability in improving the pharmacological properties of conventional drugs due to its large loading capacity, the capability to protect payload from degradation, its specific targeting, and controlled or sustained release (Kobayashi et al. 2014). Modifying the characteristic such as the nanoparticles’ size, charge, and surface coating might enhance the capability of nanoparticles in delivering drugs into target tumor through enhanced permeability and retention (EPR) effects (Matsumura and Maeda 1986; Longmire et al. 2011).

Calcium phosphate (CaP) nanoparticles have gained increasing interest in medical application because of their high biocompatibility and good biodegradability due to the fact that calcium phosphate is the inorganic mineral of human bone and teeth (Dorozhkin and Epple 2002; Rey et al. 2007). It is also not prone to microbiological degradation (Epple et al. 2010), moderately soluble at pH 7.4, and increasingly soluble below pH 6 (Tung 1998), which makes it attractive for drug delivery because CaP nanoparticles will remain intact during delivery to cells where rapid pH decrease occurs after entering endolysosome (Gerweck and Seetharaman 1996). With these advantageous characteristics of CaP nanoparticles, it is expected that we could optimize gadolinium accumulation into tumor site to achieve higher cancer cells killing effect after GdNCT treatment. We have previously shown that effective suppression of tumor growth without loss of body weight was observed on single injection of Gd-DTPA/CaP nanoparticles (Mi et al. 2015). In current work, we continued the feasibility evaluation of CaP-based nanoparticles as Gd-DTPA delivery device for GdNCT by performing assessment of antitumor effectivity on irradiated tumor-bearing mice for multiple injections of Gd-DTPA/CaP nanoparticles, in which we could achieve higher gadolinium accumulation, as a comparison with single-injected Gd-DTPA/CaP nanoparticles.

Materials and methods

Characterization of Gd-DTPA-incorporating CaP nanoparticles

An organic–inorganic nanoparticles of CaP core loaded with Gd-DTPA has been previously developed to enhance MRI contrast in solid tumors, where Gd-DTPA/CaP nanoparticles was prepared by mixing Gd-DTPA, PEG-b-PAsp, Ca2+ and HPO4 2− in buffer followed by hydrothermal synthesis at 120 °C for 20 min in Autoclave machine to increase Gd-DTPA/CaP nanoparticles stability in wet environments (Mi et al. 2014). This technique has been proven to increase the colloidal stability of CaP nanoparticles in physiological condition and enhance the relaxivity of Gd-DTPA.

For the characterization of Gd-DTPA/CaP nanoparticles, the average diameter was measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 (Malvern Instruments, UK), while the morphology of Gd-DTPA/CaP nanoparticles was confirmed using transmission electron microscope (TEM, JEM-1400, JEOL, Tokyo, Japan).

To characterize the accumulation of nanoparticles in cells, we performed calcein staining during Gd-DTPA/CaP nanoparticles preparation by adding 0.1 mM calcein, followed by purification while avoiding light. Cancer cells were seeded in 4-cm-diameter dishes at a concentration of 1 × 105 cell/ml with 2 ml DMEM medium in each well. After 24 h, the calcein-labeled Gd-DTPA/CaP nanoparticles with the concentration of 100 μM gadolinium were added to the dishes and let to incubate for another 24 h. The nuclei were then stained with Hoechst and evaluated with a LSM780 confocal scanning microscope (Carl Zeiss, Oberkochen, Germany).

Preparation of tumor-bearing mice

Colon carcinoma Col-26 cells used in this experiment were kindly supplied by National Cancer Center (Tokyo, Japan). The cells were maintained with DMEM supplemented with 10 % fetal bovine serum and then incubated in high-moisture air with 5 % CO2 at 37 °C. Female BALB/c mice were obtained from Nihon SLC (Shizuoka, Japan) and used at 6–7 weeks of age. Tumor models were prepared by subcutaneous injection of 1 × 105 of Col-26 cells into right femoral of the mice. The tumor was let to grow for two weeks until the average volume reached 100 mm3. The procedures for tumor implantation and killing of the animals were carried out following the policies of the Animal Ethics Committee of the University of Tokyo and in accordance with the Declaration of Helsinki.

Gadolinium biodistribution

We carried out two biodistribution experiments of in vivo quantitative analysis, each for single and multiple injections of Gd-DTPA/CaP nanoparticles. Gadolinium compound was injected intravenously via tail vein with the dose of 1.5 mM based on Gd-DTPA. To confirm the pharmacokinetics of Gd-DTPA/CaP nanoparticles for 0.2 ml single injection, tumor, blood, and other organ samples were harvested at 12 and 24 h following nanoparticles administration. Three times injections of Gd-DTPA/CaP nanoparticles with 10-h interval were then carried out in order to achieve higher gadolinium accumulation in tumor site. Tumor and blood samples were harvested at every 10-h interval after the first injection. Samples from both experiments were analyzed using inductively coupled plasma mass spectroscopy (ICP-MS), where volume of each organ sample was measured, followed by digestion in HNO3 left overnight, and then digested in microwave oven to ensure that all of the sample material is dissolved. The total amount of 157Gd was measured using ICP-MS, and the results were normalized to the tissue volume.

Neutron irradiation

Single- and multiple-injected mice were irradiated for 60 min at The Heavy Water Neutron Irradiation Facility of the Kyoto University Research Reactor (KUR-HWNIF) with OO-0000-F beam mode and 1 MW operating power. Mice were held within acrylic tube designed specifically for collimated neutron irradiation. Lithium fluoride shielding plate of 5 mm thickness was placed between mice holder and neutron beam to reduce neutron dose on mice body other than the tumor-bearing mice leg. Neutron fluence was measured by gold foil at 2 points around the mice leg, while gamma ray dose was measured by thermoluminescent dosimeter on the same points. Neutron irradiation was performed 24 h following single injection of Gd-DTPA/CaP nanoparticles, while neutron irradiation for the multiple-injected group was carried out at 30 h after the first administration of Gd-DTPA/CaP nanoparticles.

Evaluation of antitumor effectivity

Tumor size was measured every three days after irradiation to evaluate the antitumor activity, and the volume was calculated using the formula of V = (a × b 2)/2, where a and b are the tumor’s major and minor axes measured by a caliper, respectively. Pathological analysis was also performed for tumor samples resected on day 27 after neutron irradiation fixed on OCT compound and frozen at −80 °C. Harvested tumor samples were sliced into 6-μm sections with a cryostat and deposited on glass slide before then stained with hematoxylin and eosin (H&E). Tumor samples with the same slice thickness of 6 µm were also analyzed using apoptosis in situ detection kit Wako to detect the apoptotic cells. The kit is based on TUNEL [Terminal deoxynucleotidyl Transferase(TdT)-mediated dUTP nick end labeling] procedure, that is the addition of fluorescein—dUTP to 3′-terminals of apoptotically fragmented DNA with TdT followed by immunochemical detection using antifluorescein antibody conjugated with horseradish peroxidase (POD) and DAB as substrate. Negative control for both irradiated and non-irradiated groups was also prepared during the apoptotic assay.

Results and discussion

Gd-DTPA/CaP nanoparticles’ characterization

Monodispersity and spherical shape of Gd-DTPA/CaP nanoparticles were confirmed by TEM images as shown in Fig. 1a, with calculated volume-averaged diameter of about 60 nm (Fig. 1b). These small size nanoparticles (<100 nm) are hardly recognized by the immune system and can be easily taken up by cells. Furthermore, they are big enough to escape renal filtration, thus providing longer circulating half-life and enhanced drug accumulation in tumor tissue (Yuan 1998). No agglomeration was observed from TEM images indicating the effectivity of PEGylated surface in prohibiting interaction between each of the CaP nanoparticles as previously reported (Mi et al. 2014). The surface ξ-potential value is also confirmed to be around −0.5 mV, which is almost neutral and is preferable for drug delivery system. Positively charged nanoparticles are known to interact with negatively charged, phosphate-rich cell membrane and to be taken up in the reticuloendothelial system (RES) (Ruoslahti et al. 2010). Hence, slightly negative charged or neutral carriers are more desirable to escape from the RES. Image of calcein-stained Gd-DTPA/CaP nanoparticles shown in Fig. 1c demonstrates the accumulation of nanoparticles on the surface and inside the cancer cells, which proves the effectivity of CaP nanoparticles as carrier for Gd-DTPA. With these characteristics of Gd-DTPA/CaP nanoparticles, we could expect to achieve enhancement of gadolinium accumulation in tumor target.

Fig. 1.

Fig. 1

Open in a new tab

a TEM images of purified Gd-DTPA/CaP nanoparticles, b volume-averaged diameter distribution calculated from TEM images measured by DLS, c calcein-stained fluorescence images of Gd-DTPA/CaP nanoparticles indicating its accumulation on the surface and into cancer cells

Gadolinium biodistribution

Free Gd-DTPA was reported to be rapidly cleared from plasma and did not accumulate in tumor tissue, and that only trace amounts of gadolinium (nearly 0.001 % dose/ml) were found in plasma 1 h after intravenous administration, while CaP nanoparticles have been previously proven to be capable of extending Gd-DTPA blood circulation time, which then increased gadolinium accumulation into tumor site (Mi et al. 2014).

Quantitative analysis from ICP-MS measurement results of gadolinium accumulation in tumor and several mice organs is shown in Table 1, where we could observe tumor-to-blood (T/B) ratio of around 2.4 at 24 h after Gd-DTPA/CaP nanoparticles injection. This number is comparable to T/B ratio for BNCT, where the optimum ratio that could be reached is considered to be around 3–4 (Barth et al. 2005). Small accumulation and fast clearance of gadolinium observed in brain samples are the evidence of blood brain barrier existence in normal brain because of no modification in Gd-DTPA/CaP nanoparticles for the disruption of blood brain barrier.

Table 1.

Gadolinium accumulation in tumor and several organs of tumor-bearing mice at 12 and 24 h after single injection of Gd-DTPA/CaP nanoparticles

Gd concentration (ug/g or mL)
12 h 24 h
Tumor 5.85 ± 0.64 8.03 ± 0.82
Blood 14.55 ± 0.50 3.29 ± 0.40
Liver 10.71 ± 0.17 16.71 ± 0.60
Spleen 7.08 ± 0.41 10.03 ± 0.30
Kidney 2.66 ± 0.24 2.13 ± 0.06
Brain 0.16 ± 0.01 0.04 ± 0.01
Open in a new tab

Nevertheless, the uptake of gadolinium by liver, kidney, and spleen after compound injection was still quite high, probably because of its characteristics that circulates in blood at first and then distributes into the interstitial space or is eliminated by the kidneys in the same manner with the results reported by Weinmann et al. (1984) that major portion was discovered in liver and spleen as the results of predominate renal elimination. Therefore, it is necessary to ensure minimum neutron dose into organs other than tumor site to secure tolerable irradiation effect on normal tissue. However, the ratio of gadolinium concentration in tumor to those in kidney was much smaller compared to our previous result with gadoteridol-encapsulating liposome (Dewi et al. 2013), proving the effectivity of Gd-DTPA/CaP nanoparticles in escaping renal filtration with its small particle size.

Higher gadolinium accumulation in tumor site was successfully achieved for multiple injections of Gd-DTPA/CaP nanoparticles as shown in Fig. 2a, where gadolinium concentration reached the amount of more than three times higher compared to those at 10 h after the first injection. Significant increase in gadolinium concentration in blood plasma was also observed at 30 h after the first injection (Fig. 2b). This result indicates the prolonged blood circulation of Gd-DTPA/CaP nanoparticles, which is important because with repeated passages of the delivery system through the tumor microvascular bed, a greater efficiency of extravasations per unit volume of the transports could be achieved.

Fig. 2.

Fig. 2

Open in a new tab

Gadolinium biodistribution for mice with multiple injections of Gd-DTPA/CaP nanoparticles, a in tumor site, b in blood plasma

Neutron irradiation and antitumor effectivity

Tumor volume growth by time is shown in Fig. 3, where we could observe that Gd-DTPA/CaP-irradiated group revealed up to four times tumor growth suppression compared to non-treated group. Also, no significant weight loss was observed after neutron irradiation and all mice survived until the end of observation, suggesting low toxicity of Gd-DTPA/CaP nanoparticles for Gd-NCT. However, the multiple-injected mice group did not show better tumor growth suppression even though gadolinium concentration accumulated in tumor site is much higher compared to the single-injected mice group. There might be a possibility that neutron depression occurred in this group and that the neutron was being absorbed and could not reach deeper part of the tumor, which might reduce the effectivity of cancer cells killing. Nevertheless, we could still observe tumor growth suppression after neutron irradiation, which suggests the possibility of cancer cells killing from secondary particles of GdNCR.

Fig. 3.

Fig. 3

Open in a new tab

Tumor growth suppression evaluated until 27 days after neutron irradiation for both single and multiple injections of Gd-DTPA/CaP nanoparticles (p < 0.08 compared to non-irradiated group)

During the 60 min of neutron irradiation, average neutron fluence was measured to be 1.8 × 1012 n/cm2 for thermal neutron range and 3.2 × 1011 n/cm2 for epithermal neutron, while physical dose from neutron and gamma ray measurement results is shown in Table 2. It is defined that thermal neutron region is below 0.5 keV, the epithermal neutron region is 0.5–10 keV, and the fast neutron region is over 10 keV. Even though the measured physical dose for 157Gd was lower compared to the dose contribution from other components, the limitation of this value needs to be taken into account since a more pertinent equivalent dose calculation is necessary to express cancer cells killing effect after GdNCT treatment, where the Auger and Coster-Kronig electron might contribute significant biological effect in killing the cancer cells.

Table 2.

Average physical dose from the measurement of gold foil and TLD during neutron irradiation

Physical dose (Gy)
Thermal neutron (~0.5 eV) 2.4 × 10−1
Epithermal neutron (0.5–10 keV) 2.55 × 10−2
Fast neutron (10 keV~) 1.80 × 10−1
Gamma ray 4.70 × 10−1
Total 9.10 × 10−1
Gadolinium-157 (1 ppm) 1.4 × 10−1
Open in a new tab

Among GdNCR products, Auger electrons are the most biologically relevant to be comparable with high LET alpha particle and lithium ions from BNCR. They have energy range between 0 and 50 keV with an average of 4.19 keV (Goorley and Nikjoo 2000), with corresponding average LET of 0.3 MeV/mm. By comparison, the average LET in BNCR is 0.2 MeV/mm for both lithium and alpha particles (De Stasio et al. 2005). Contribution of Auger electrons to high-resolution neutron radiographs with gadolinium converter plate was already reported by (Harms and Norman 1972), providing the evidence for the existence of Auger electrons during GdNCR. The presence of Auger electrons after GdNCR has also been proven with the range of no more than 2.7 mg/cm2 (Shih and Brugger 1992). Yasui et al. (2008) have also reported the effectivity of gadolinium neutron capture therapy in vitro on glioblastoma multiforme cells, where they observed significant enhancement of cell killing on cells preloaded with Gd-DTPA and treated with neutron irradiation. Following observation of no necrosis with high cancer cells, killing effectivity after GdNCT was reported in their experiment compared to other mice group treated with gamma, fast neutrons, and modified enhanced thermal neutron beam (Yasui and Owens 2012). They referred that the discovery of extreme autophagy in GdNCT-treated group might provide the clues in understanding how Auger electron irradiation kills the cancer cells. These short-range products may enhance the therapeutic effects of gadolinium neutron capture therapy (GdNCT) and are expected to deliver high cell killing effect when incorporated into DNA (Kassis et al. 1982), and with proper techniques, GdNCT might be made competitive to BNCT.

The therapeutic effects of GdNCT were also observed from pathological analysis results of tumor cells as shown by H&E staining results in Fig. 4, where the nucleus and cytoplasm of tumor cells treated with Gd-DTPA/CaP nanoparticles were destroyed after GdNCT treatment, while the non-irradiated group showed normal histology with survived nuclei and abundant cytoplasm, demonstrating the characteristics of proliferative tumor cells. Similar H&E result was observed between single-injected and multiple-injected groups, which agrees with the results from tumor growth suppression effect. These results are also supported by TUNEL assay shown in Fig. 5, where the number of cells stained (brown-colored cells), which correlates with the number of apoptosis, was higher on GdNCT-treated groups compared to the non-irradiated ones, in which we observed that most of the cells were unstained.

Fig. 4.

Fig. 4

Open in a new tab

Pathological analysis results from H&E staining of tumor cells after GdNCT. Original magnification ×200, a Gd-DTPA/CaP nanoparticles single-injected irradiated group, b Gd-DTPA/CaP nanoparticles multiple-injected irradiated group, c Gd-DTPA/CaP nanoparticles single-injected non-irradiated group, d DTPA/CaP nanoparticles multiple-injected non-irradiated group

Fig. 5.

Fig. 5

Open in a new tab

TUNEL staining of tumor slice after Gd-NCT. Original magnification ×200, a Gd-DTPA/CaP nanoparticles single-injected irradiated group, b Gd-DTPA/CaP nanoparticles multiple-injected irradiated group, c Gd-DTPA/CaP nanoparticles single-injected non-irradiated group, d DTPA/CaP nanoparticles multiple-injected non-irradiated group

Conclusion

In this work, we have performed antitumor effectivity evaluation for both single and multiple injections of Gd-DTPA/CaP nanoparticles in GdNCT experiment. Gadolinium concentration accumulated in tumor site for single-injected group was still considered quite low for NCT treatment, considering the optimal 157Gd concentration in tumors for Gd-NCT was reported to be 50–200 μg/g tumor tissues and less than 1000 ppm since neutron fluence rapidly decrease in the deeply seated tumor due to high absorption of neutron by gadolinium atoms (Le and Cui 2006). Nevertheless, we could still observe tumor growth suppression after neutron irradiation as well as cancer cells killing effect indicating the possible effectivity of GdNCT. Higher gadolinium accumulation was successfully achieved with multiple injections of Gd-DTPA/CaP nanoparticles. However, we could not observe better tumor growth suppression after GdNCT treatment, which indicates the possibility of neutron depression on mice group with higher concentration of gadolinium. Even though similar results on single-injected and multiple-injected group were also observed on H&E and TUNEL staining, further investigation is necessary to confirm the reason of moderate tumor growth suppression in mice group with multiple injections of Gd-DTPA/CaP nanoparticles.

Funding

This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan (Nos. 25670571, and 24390311 to Hironobu Yanagie).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The procedures for tumor implantation and killing of the animals were carried out following the policies of the Animal Ethics Committee of the University of Tokyo and in accordance with the Declaration of Helsinki.

References

  1. Aime S, Caravan P (2009) Biodistribution of gadolinium-based contrast agents, including gadolinium deposition. J Magn Reson Imaging 30(6):1259–1267 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barth RF, Coderre JA, Vicente MGH, Blue TE (2005) Boron neutron capture therapy of cancer: current status and future prospects. Clin Cancer Res 11:3987–4002 [DOI] [PubMed] [Google Scholar]
  3. Brugger RM, Shih JA (1989) Evaluation of gadolinium-157 as a neutron capture therapy agent. Strahlenther Onkol 165:153–156 [PubMed] [Google Scholar]
  4. Cerullo N, Bufalino D, Daquino G (2009) Progress in the use of gadolinium for NCT. Appl Radiat Isot 67:S157–S160 [DOI] [PubMed] [Google Scholar]
  5. Chadha M, Capala J, Coderre JA, Elowitz EH, Iwai J, Joel DD, Liu HB, Wielopolski L, Chanana AD (1998) Boron neutron-capture therapy (BNCT) for glioblastoma multiforme (GBM) using the epithermal neutron beam at the Brookhaven National Laboratory. Int J Radiat Oncol Biol Phys 40(4):829–834 [DOI] [PubMed] [Google Scholar]
  6. De Stasio G, Casalbore P, Pallini R et al (2001) Gadolinium in human glioblastoma cells for gadolinium neutron capture therapy. Cancer Res 61:4272–4277 [PubMed] [Google Scholar]
  7. De Stasio G et al (2005) Are gadolinium contrast agents suitable for gadolinium neutron capture therapy? Neurol Res 27:387–398 [DOI] [PubMed] [Google Scholar]
  8. Dewi N, Yanagie H, Zhu H et al (2013) Tumor growth suppression by gadolinium–neutron capture therapy using gadolinium-entrapped liposome as gadolinium delivery agent. Biomed Pharmacother 67:451–457 [DOI] [PubMed] [Google Scholar]
  9. Dorozhkin SV, Epple M (2002) Biological and medical significance of calcium phosphate. Angew Chem Int Ed Engl 41(17):3130–3146 [DOI] [PubMed] [Google Scholar]
  10. Epple M et al (2010) Application of calcium phosphate nanoparticles in biomedicine. J Mater Chem 20:18–23 [Google Scholar]
  11. Fuwa N, Suzuki M, Sakurai Y et al (2008) Treatment results of boron neutron capture therapy using intra-arterial administration of boron compounds for recurrent head and neck cancer. Br J Radiol 81:749–752 [DOI] [PubMed] [Google Scholar]
  12. Gerweck LE, Seetharaman K (1996) Cellular pH gradient in tumor versus normal tissue. Cancer Res 56:1194–1198 [PubMed] [Google Scholar]
  13. Goorley T, Nikjoo H (2000) Electron and photon spectra for three gadolinium-based cancer therapy approaches. Radiat Res 154:556–563 [DOI] [PubMed] [Google Scholar]
  14. Hambley TW, Hait WN (2009) Is anticancer drug development heading in the right direction? Cancer Res 69:1259–1262 [DOI] [PubMed] [Google Scholar]
  15. Harms AA, Norman GR (1972) The role of internal conversion electrons in gadolinium-exposure neutron imaging. J Appl Phys 43(7):3209–3212 [Google Scholar]
  16. Henriksson R, Capala J, Michanek A et al (2008) Boron neutron capture therapy (BNCT) for glioblastoma multiforme: a phase II study evaluating a prolonged high dose of boronophenylalanine (BPA). Radiother Oncol 88:183–191 [DOI] [PubMed] [Google Scholar]
  17. Ichikawa H, Uneme T, Andoh T, Arita Y, Fujimoto T, Suzuki M et al (2014) Gadolinium-loaded chitosan nanoparticles for neutron-capture therapy: influence of micrometric properties of the nanoparticles on tumor-killing effect. Appl Radiat Isot 88:109–113 [DOI] [PubMed] [Google Scholar]
  18. Joensuu H, Kankaanranta L, Seppälä T et al (2003) Boron neutron capture therapy of brain tumors: clinical trials at the Finnish facility using boronophenylalanine. J Neurooncol 62:123–134 [DOI] [PubMed] [Google Scholar]
  19. Kassis AI, Adelstein SJ, Haydock C, Sastry KSR, McElvany KD, Welch MJ (1982) Lethality of Auger electrons from the decay of bromine-77 in the DNA of mammalian cells. Radiat Res 90:362–373 [PubMed] [Google Scholar]
  20. Kobayashi H, Watanabe R, Choyke PL (2014) Improving conventional enhanced permeability and retention (EPR) effects; What is the appropriate target? Theranostics 4(1):81–89 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Le UM, Cui Z (2006) Long-circulating gadolinium-encapsulated liposomes for potential application in tumor neutron capture therapy. Int J Pharm 312:105–112 [DOI] [PubMed] [Google Scholar]
  22. Locher GL (1936) Biological effects and therapeutic possibilities of neutrons. Am J Roentgenol Radium Ther 36:1–13 [Google Scholar]
  23. Longmire MR, Ogawa M, Choyke PL, Kobayashi H (2011) Biologically optimized nanosized molecules and particles: more than just size. Bioconjug Chem 22:993–1000 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Martin RF, D’Cunha G, Pardee M et al (1989) Induction of DNA doublestrand breaks by 157Gd neutron capture. Pigment Cell Res 2:330–332 [DOI] [PubMed] [Google Scholar]
  25. Masiakowski JT, Horton JL, Peters LJ (1992) Gadolinium neutron capture therapy for brain tumors: a computer study. Med Phys 19:1277–1284 [DOI] [PubMed] [Google Scholar]
  26. Matsumura Y, Maeda H (1986) A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the anti-tumor agent smancs. Cancer Res 46:6387–6392 [PubMed] [Google Scholar]
  27. Mi P, Kokuryo D, Cabral H, Kumagai M, Nomoto T, Aoki I, Terada Y, Kishimura A, Nishiyama N, Kataoka K (2014) Hydrothermally synthesized PEGylated calcium phosphate nanoparticles incorporating Gd-DTPA for contrast enhanced MRI diagnosis of solid tumors. J Controlled Release 174:63–71 [DOI] [PubMed] [Google Scholar]
  28. Mi P, Dewi N, Yanagie H, Kokuryo D, Suzuki M, Sakurai Y, Li Y, Aoki I, Ono K, Takahashi H, Cabral H, Nishiyama N, Kataoka K (2015) Hybrid calcium phosphate-polymeric micelles incorporating gadolinium chelates for imaging-guided gadolinium neutron capture tumor therapy. ACS Nano. doi:10.1021/acsnano.5b00532 [DOI] [PubMed] [Google Scholar]
  29. Rey C, Combes C, Drouet C, Sfihi H, Barroug A (2007) Physico-chmeical properties of nanocrystalline apatites: implications for biominerals and biomaterials. Mater Sci Eng 27:198–205 [Google Scholar]
  30. Ruoslahti E, Sangeeta NB, Michael JS (2010) Targeting of drugs and nanoparticles to tumor. J Cell Biol 188(6):759–768 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shih JL, Brugger RM (1992) Gadolinium as neutron capture therapy agent. Med Phys 19(3):733–744 [DOI] [PubMed] [Google Scholar]
  32. Tokumitsu H et al (2000) Gadolinium neutron-capture therapy using novel gadopentetic acid-chitosan complex nanoparticles: in vivo growth suppression of experimental melanoma solid tumor. Cancer Lett 150:177–182 [DOI] [PubMed] [Google Scholar]
  33. Tung M (1998) Calcium phosphates: structure, composition, solubility, and stability. In: Amjad Z (ed) Calcium phosphates in biological and industrial systems. Kluwer Academic Publishers, Boston, pp 1–19 [Google Scholar]
  34. Watanabe T, Ichikawa H, Fukumori Y (2002) Tumor accumulation of gadolinium in lipid-nanoparticles intravenously injected for neutron-capture therapy of cancer. Eur J Pharm Biopharm 54:119–124 [DOI] [PubMed] [Google Scholar]
  35. Weinmann HJ, Brasch RC, Press WR, Wesbey GE (1984) Characteristics of gadolinium-DTPA complex: a potential NMR contrast agent. Am J Roentgenol 142:619–624 [DOI] [PubMed] [Google Scholar]
  36. Yamamoto T, Matsumura A, Nakai K, Shibata Y, Endo K, Sakurai F, Kishi T, Kumada H, Yamamoto K, Torii Y (2004) Current clinical results of the Tsukuba BNCT trial. Appl Radiat Isot 61(5):1089–1093 [DOI] [PubMed] [Google Scholar]
  37. Yanagie H, Tomita T, Kobayashi H, Fujii Y, Takahashi T, Hasumi K, Nariuchi H, Sekiguchi M (1991) Application of boronated anti-CEA immunoliposome to tumour cell growth inhibition in in vitro boron neutron capture therapy model. Br J Cancer 63(4):522–526 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yanagie H, Ogata A, Sugiyama H, Eriguchi M, Takamoto S, Takahashi H (2008) Application of drug delivery system to boron neutron capture therapy for cancer. Expert Opin Drug Deliv 5(4):427–443 [DOI] [PubMed] [Google Scholar]
  39. Yasui L, Owens K (2012) Necrosis is not induced by gadolinium neutron capture in glioblastoma multiforme cells. Int J Radiat Biol 88:980–990 [DOI] [PubMed] [Google Scholar]
  40. Yasui L et al (2008) Gadolinium neutron capture in glioblastoma multiforme cells. Int J Radiat Biol 84:1130–1139 [DOI] [PubMed] [Google Scholar]
  41. Yuan F (1998) Transvascular drug delivery in solid tumors. Semin Radiat Oncol 8:164–175 [DOI] [PubMed] [Google Scholar]

Articles from Journal of Cancer Research and Clinical Oncology are provided here courtesy of Springer

RESOURCES