Improved Boron Neutron Capture Therapy Using Integrin αvβ3-Targeted Long-Retention-Type Boron Carrier in a F98 Rat Glioma Model

Simple Summary The development of a novel boron carrier is a key step in clinical boron neutron capture therapy (BNCT). We previously reported that maleimide-functionalized closo-dodecaborate albumin conjugate (MID-AC) with albumin as the drug delivery system is an effective boron carrier for the F98 glioma-bearing rat brain tumor model. In this study, the efficacy of BNCT with cRGD-MID-AC, a cyclic arginine-glycine-aspartate (cRGD) targeting integrin αvβ3 added to MID-AC, was evaluated in a glioma-bearing rat brain tumor model. Although the cellular boron concentration of cRGD-MID-AC was lower than that of boronophenylalanine (BPA), in vitro neutron-irradiation experiments confirmed that the cell-killing effect of BNCT using cRGD-MID-AC was similar to that of BNCT using BPA. In vivo biodistribution showed a sufficient boron concentration in the tumor after intravenous administration. In neutron-irradiation experiments, the BNCT group using cRGD-MID-AC showed significantly prolonged survival compared to the untreated group, and long-term survivors were observed. This drug shows promise as a novel agent for BNCT. Abstract Integrin αvβ3 is more highly expressed in high-grade glioma cells than in normal tissues. In this study, a novel boron-10 carrier containing maleimide-functionalized closo-dodecaborate (MID), serum albumin as a drug delivery system, and cyclic arginine-glycine-aspartate (cRGD) that can target integrin αvβ3 was developed. The efficacy of boron neutron capture therapy (BNCT) targeting integrin αvβ3 in glioma cells in the brain of rats using a cRGD-functionalized MID-albumin conjugate (cRGD-MID-AC) was evaluated. F98 glioma cells exposed to boronophenylalanine (BPA), cRGD-MID-AC, and cRGD + MID were used for cellular uptake and neutron-irradiation experiments. An F98 glioma-bearing rat brain tumor model was used for biodistribution and neutron-irradiation experiments after BPA or cRGD-MID-AC administration. BNCT using cRGD-MID-AC had a sufficient cell-killing effect in vitro, similar to that with BNCT using BPA. In biodistribution experiments, cRGD-MID-AC accumulated in the brain tumor, with the highest boron concentration observed 8 h after administration. Significant differences were observed between the untreated group and BNCT using cRGD-MID-AC groups in the in vivo neutron-irradiation experiments through the log-rank test. Long-term survivors were observed only in BNCT using cRGD-MID-AC groups 8 h after intravenous administration. These findings suggest that BNCT with cRGD-MID-AC is highly selective against gliomas through a mechanism that is different from that of BNCT with BPA.

experiments to measure the retention rate of boron, a cell culture medium with 10 µg B/mL of BPA, cRGD-MID-AC, or cRGD + MID was incubated for 24 h. Then, the medium was exchanged for a boron-free medium and incubated for 1, 6, or 24 h. The medium with the boron carriers or the boron-free medium was removed, and the cells were washed twice with 4% phosphate-buffered saline (PBS), detached by trypsin-ethylenediamine tetraacetic acid solution, and all the cells in the dish were collected. The culture medium was then added in the dish, and the cells were counted after centrifugation twice (at 200× g for 5 min). The cells were then dissolved overnight in a 1 N nitric acid solution (Wako Pure Chemical Industries, Osaka, Japan), and the amount of intracellular boron was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES; iCAP6300 emission spectrometer, Hitachi, Tokyo, Japan). The intracellular boron concentrations were defined as µg boron (B)/10 9 cells.

In Vitro Neutron-Irradiation Experiments
The cytotoxicity of each boron carrier in BNCT was evaluated using colony-forming assays. F98 glioma cells were used for the in vitro neutron-irradiation experiments. They were incubated in 150 cm 2 tissue culture flasks with 20 mL of each medium for four groups: group 1, neutron only (boron-free medium); group 2, BPA; group 3, cRGD-MID-AC; and group 4, cRGD + MID. All boron-exposure groups were exposed for 2.5 h to 10 µg B/mL of boron-containing medium derived from each drug. Neutron irradiation was then performed. Cells were irradiated for 10, 20, and 30 min at a reactor power of 1 MW with a neutron flux of 1.1 × 10 9 neutrons/cm 2 /s at KURNS. After neutron irradiation, cells from each sample were collected and counted. The cell solution was diluted to the predetermined number of cells, and the cells were seeded in a 60 mm dish (Becton, Dickson, and Company, Franklin Lakes, NJ, USA) (Three dishes were prepared per group and per number of cells). The cells were then incubated for seven days. Finally, they were fixed with 90% ethanol and stained with Giemsa. The survival fraction (SF) was calculated by counting the number of colonies consisting of more than 50 cells and dividing it by the number of colonies of the control group. In this experiment, the RBE (relative biological effectiveness) of the neutron beam and the CBE (compound biological effectiveness) of each boron carrier were estimated by considering the linear-quadratic (LQ) model obtained from the X-ray irradiation of F98 glioma cells and the physical dose to achieve SF = 0.1 in each group [39].

Biodistribution of Boron in the F98 Glioma-Bearing Rats after Intravenous Administration of Each Boron Carrier
Approximately 12-14 days after implantation of 10 5 F98 glioma cells, when the tumor was expected to have grown sufficiently, each boron carrier was administered at 12 mg boron (B)/kg body weight (b.w.). At each predetermined time, the rats were euthanized, and each tissue (the tumor, brain, blood, heart, lung, liver, kidney, spleen, skin, and muscle samples) was removed. Each organ was weighed and digested with 1 N nitric acid solution after weighing. The amount of boron in each organ was measured by ICP-AES. All results (boron concentrations) were defined as µg boron (B)/g.
All rats were anesthetized through an intraperitoneal injection of the anesthetics, and BPA or cRGD-MID-AC was administered to the assigned experimental groups. Only their heads were irradiated with neutrons at the KURNS. They were irradiated for 20 min at a reactor power of 5 MW and a neutron flux of 9.6 × 10 8 neutrons/cm 2 /s at the Heavy Water Irradiation Facility at KURNS. All rats were observed until death or euthanasia. In addition, the therapeutic effects were evaluated by Kaplan-Meier survival curves, and the percent increase in life span (%ILS) was calculated by the following equation: (the median survival times; MST of each BNCT group-MST of untreated group) × 100/(MST of untreated).

Estimated Physical Dose and Biologically Photon-Equivalent Dose
The physical dose was determined using the dose calculated from the thermal, epithermal, fast neutron, and gamma rays of the irradiated neutrons. The equation D B + D N + D H + D γ was used, and each factor corresponded to 10 B(n,α) 7 Li, 14 N(n,p) 14 C, and 1 H(n,n) 1 H capture reactions and γ-rays, respectively. What D B , D N , D H , and D γ mean and how to calculate them have already reported, and the physical doses to the brain and brain tumors were calculated for each group and calculated according to [32,36]. The estimated photonequivalent dose was calculated by the following equation: D B × CBE + D N × relative biological effectiveness (RBE N ) + D H × relative biological effectiveness (RBE H ) + D γ . Both the physical doses and estimated photon-equivalent doses were corrected according to the previous report [32,36]. CBE refers to the biological effectiveness ratio in boron neutron capture reaction that is specific to the irradiated tissue or boron carrier. In the previous report, the CBE factor for normal brain was defined as 1.35 in BNCT with BPA [2,40]. In order to calculate the estimated photon-equivalent doses for in vivo irradiation experiment, the CBE for BPA and cRGD-MID-AC was calculated by the results obtained in the in vitro experiment.

Statistical Analysis
In the in vitro cellular uptake experiments, the intracellular boron concentrations in all cell lines were evaluated by the Student's t-test. Survival times were evaluated by Kaplan-Meier curves. Log-rank tests were used to determine significant differences between the groups. For all tests, statistical p-values of less than 0.05 were evaluated as a significant difference. All the results were analyzed using JMP ® Pro version 15.1.0. software (SAS, Cary, NC, USA).

In Vitro Cellular Boron Uptake Experiments in F98, C6 Glioma, and 9L Gliosarcoma Cells
The boron concentrations in F98, C6 glioma, and 9L gliosarcoma cells after exposure to each boron carrier from 1 h to 24 h are shown in Figure 1A-C. The retention rates of boron for BPA, cRGD-MID-AC, and cRGD + MID are shown in Figure 1D-F. The cellular boron concentrations of each cell at 1, 6, and 24 h after exposure to 10 µg B/mL of each boron carrier and at 1 (24 + 1), 6 (24 + 6), and 24 (24 + 24) h after 1, 6, and 24 h of additional incubation, when the medium was changed to boron-free, are shown in Tables A1-A3. The boron concentrations of BPA and cRGD-MID-AC from 1 to 24 h in all cells gradually increased over time. In contrast, the boron concentrations of cRGD + MID were the highest at 6 h and did not show significant differences until 24 h later in all cells ( Figure 1A-C). The boron concentration of BPA from 1 h to 24 h in all cell lines was significantly higher than that of cRGD-MID-AC and cRGD + MID (p < 0.05). However, from 24 + 1 h to 24 + 24 h, the boron concentration of cRGD-MID-AC in all cell lines was significantly higher than that of BPA (p < 0.05, except 9L 24 + 24 h). The retention rates of boron for BPA, cRGD-MID-AC, and cRGD + MID after 1 h of additional incubation when the medium was changed to boron-free were 14.7%, 74.6%, and 65.2% in F98 glioma cells; 15.7%, 88.8%, and 78.4% in C6 glioma cells; and 23.3%, 85.6%, and 59.1% in 9L rat gliosarcoma cells, respectively ( Figure 1D-F). After 6 and 24 h of additional incubation, the retention rates of boron for BPA, cRGD-MID-AC, and cRGD + MID were 12.0%, 72.3%, and 56.4% (6 h) and 9.5%, 34 Figure 1D-F). The concentration of boron retained by tumor cells in each group decreased rapidly in BPA after replacement with boron-free medium, whereas it decreased gradually over time in all the other groups. changed to boron-free were 14.7%, 74.6%, and 65.2% in F98 glioma cells; 15.7%, 88.8%, and 78.4% in C6 glioma cells; and 23.3%, 85.6%, and 59.1% in 9L rat gliosarcoma cells, respectively ( Figure 1D-F). After 6 and 24 h of additional incubation, the retention rates of boron for BPA, cRGD-MID-AC, and cRGD + MID were 12.0%, 72.3%, and 56.4% (6 h) and 9.5%, 34.3%, and 21.1% (24 h) in F98 glioma cells; 15.6%, 82.8%, and 75.3% (6 h) and 7.4%, 26.5%, and 22.6%(24 h) in C6 glioma cells; and 16.3%, 70.7%, and 54.3% (6 h) and 12.7%, 31.3%, and 24.7% (24 h) in 9L rat gliosarcoma cells, respectively ( Figure 1D-F). The concentration of boron retained by tumor cells in each group decreased rapidly in BPA after replacement with boron-free medium, whereas it decreased gradually over time in all the other groups.

Neutron Irradiation in the In Vitro Experiments
The results of the in vitro neutron-irradiation experiments are shown in Figure 2

Neutron Irradiation in the In Vitro Experiments
The results of the in vitro neutron-irradiation experiments are shown in Figure  Using the linear-quadratic (LQ) model obtained from X-ray irradiation of F98 glioma cells, the doses to calculate the CBE factors for the BPA, cRGD-MID-AC, and cRGD + MID groups were estimated from the colony-forming assays. The physical doses required to achieve SF = 0.1 in the BPA, cRGD-MID-AC, and cRGD + MID groups were 0.75, 0.85, and 1.48 Gy, respectively. (In the LQ model, the corresponding dose was 6.45 Gy [36].) The CBE factors of the BPA, cRGD-MID-AC, and cRGD + MID groups (determined by the calculated RBE) were 2.69, 2.26, and 0.75, respectively.

Biodistribution of Boron in the F98 Glioma-Bearing Rats after Intravenous Administration of Each Boron Carrier
Boron concentrations in each organ were evaluated at 2.5, 8, and 24 h after intravenous administration of cRGD-MID-AC or BPA. In the case of cRGD-MID-AC, the boron concentrations in the tumor were 10.1 ± 1.6 (0.8 ± 0.2 in the brain and 41.6 ± 5.6 in the blood), 17.0 ± 1.8 (0.9 ± 0.1 in the brain and 40.3 ± 8.4 in the blood), and 13.1 ± 1.9 (0.7± 0.1 in the brain and 17.7 ± 2.3 in the blood) µg B/g at 2.5, 8, and 24 h, respectively. The boron concentration in the tumor was the highest at 8 h after intravenous administration of cRGD-MID-AC and tended to be retained for a longer period of 24 h. In contrast, in the case of BPA, the boron concentrations were 20.6 ± 2.2 (5.5 ± 0.6 in the brain and 7.7 ± 0.5 in the blood), 18.2 ± 2.9 (5.3 ± 0.5 in the brain and 4.8 ± 0.3 in the blood), and 8.2 ± 0.8 (2.3 ± 0.3 in the brain and 2.9 ± 0.4 in the blood) µg B/g at 2.5, 8, and 24 h, respectively [16]. The boron concentrations in the tumor were the highest 2.5 h after intravenous administration of BPA and decreased gradually. In addition, after every hour, the tumor/normal brain ratio of cRGD-MID-AC was much higher than that of BPA. Table 1 presents a summary of our results, and Figure 3 shows the boron concentrations in each organ and the ratio of tumor to normal brain tissue (T/Br ratio).

Biodistribution of Boron in the F98 Glioma-Bearing Rats after Intravenous Administration of Each Boron Carrier
Boron concentrations in each organ were evaluated at 2.5, 8, and 24 h after intravenous administration of cRGD-MID-AC or BPA. In the case of cRGD-MID-AC, the boron concentrations in the tumor were 10.1 ± 1.6 (0.8 ± 0.2 in the brain and 41.6 ± 5.6 in the blood), 17.0 ± 1.8 (0.9 ± 0.1 in the brain and 40.3 ± 8.4 in the blood), and 13.1 ± 1.9 (0.7± 0.1 in the brain and 17.7 ± 2.3 in the blood) µg B/g at 2.5, 8, and 24 h, respectively. The boron concentration in the tumor was the highest at 8 h after intravenous administration of cRGD-MID-AC and tended to be retained for a longer period of 24 h. In contrast, in the case of BPA, the boron concentrations were 20.6 ± 2.2 (5.5 ± 0.6 in the brain and 7.7 ± 0.5 in the blood), 18.2 ± 2.9 (5.3 ± 0.5 in the brain and 4.8 ± 0.3 in the blood), and 8.2 ± 0.8 (2.3 ± 0.3 in the brain and 2.9 ± 0.4 in the blood) µg B/g at 2.5, 8, and 24 h, respectively [16]. The boron concentrations in the tumor were the highest 2.5 h after intravenous administration of BPA and decreased gradually. In addition, after every hour, the tumor/normal brain ratio of cRGD-MID-AC was much higher than that of BPA. Table 1 presents a summary of our results, and Figure 3 shows the boron concentrations in each organ and the ratio of tumor to normal brain tissue (T/Br ratio).  Table 1. Boron concentrations in the tumor, brain (normal brain), and blood after intravenous administration of each boron carrier in F98 glioma-bearing rats.

Boron Carrier a Time b (h) n c Boron Concentration ± SD (µg B/g) d Ratio
Tumor

Survival Analysis of the In Vivo Neutron-Irradiation Experiments
Neutron-irradiation experiments were performed at both 2.5 and 8 h because the boron concentration in the tumor was the highest at 2.5 h for BPA and at 8 h for cRGD-MID-AC according to the biodistribution experiments. The treatment effect was evaluated using Kaplan-Meier curves ( Figure 4A,B). Each MST and %ILS value is shown in Table 2.
Statistically significant differences were observed between the untreated group and all BNCT groups evaluated by the log-rank test ( Table 2). Ninety days after the F98 glioma cell implantation, one F98 glioma-bearing rat survived for a long time only in the group of BNCT using cRGD-MID-AC 8 h.

Estimation of Physical and Biologically Photon-Equivalent Doses
The physical and photon-equivalent doses for the brain and tumor by neutronirradiation experiments were calculated using the CBE factor of each boron carrier obtained as a reference and in vitro neutron-irradiation experiment and the mean boron concentrations in the tumor obtained from in vivo biodistribution experiments. RBE N and RBE H were adopted as 3.0 according to the previous report [41]. The calculated photon-equivalent doses for the brain tumor obtained with BNCT using BPA at 2.5 h and 8 h were 10.9 Gy-Eq and 9.9 Gy-Eq, respectively, and with BNCT using cRGD-MID-AC at 2.5 h and 8 h were 5.8 Gy-Eq and 9.5 Gy-Eq, respectively (Table 3).

Survival Analysis of the In Vivo Neutron-Irradiation Experiments
Neutron-irradiation experiments were performed at both 2.5 and 8 h because the boron concentration in the tumor was the highest at 2.5 h for BPA and at 8 h for cRGD-MID-AC according to the biodistribution experiments. The treatment effect was evaluated using Kaplan-Meier curves ( Figure 4A,B). Each MST and %ILS value is shown in Table 2.
Statistically significant differences were observed between the untreated group and all BNCT groups evaluated by the log-rank test ( Table 2). Ninety days after the F98 glioma cell implantation, one F98 glioma-bearing rat survived for a long time only in the group of BNCT using cRGD-MID-AC 8 h.      In the case of BPA, the CBE factor for the normal brain tissue was 1.35. * In the case of cRGD-MID-AC, the CBE for normal brain tissue was unknown. Therefore, the boxes are kept blank.

Discussion
cRGD-MID-AC was adapted for BNCT against an experimental brain tumor model for the first time. A significant difference between untreated and BNCT using cRGD-MID-AC was observed by log-rank test, which suggested that BNCT using cRGD-MID-AC is effective in the experimental brain tumor model ( Figure 4A,B, and Table 2). In addition, long-term survivors were observed in BNCT using cRGD-MID-AC for 8 h, whereas no long-term-surviving individuals were observed in the other groups. This result suggested that cRGD-MID-AC accumulates in glioma cells with high integrin expression. In other words, the effect of cRGD-MID-AC may have been pronounced in the F98 glioma-bearing rats with high integrin expression [42].
Although MID-AC accumulates in tumor cells and can prolong the boron concentration in the tumor for up to 24 h after intravenous administration, the boron concentration delivered to the brain tumor by MID-AC was still low (<8.5 µg B/g) [16]. Thus, MID-AC has been modified by conjugation with cRGD, which binds strongly to integrins, especially integrin α v β 3 , and this conjugate was thought to contribute to improving the efficacy of BNCT against gliomas. Integrins are a family of cell-cell and cell-extracellular matrix adhesion molecules [43]. Among them, integrin α v β 3 is overexpressed in glioma cells, whereas its expression in normal cells is low and is related to invasion, proliferation, and angiogenesis of tumor cells [20][21][22][23][43][44][45]. The CENTRIC study (phase III trial), which investigated the efficacy of cilengitide, a selective integrin α v β 3 inhibitor, as antitumor therapy against glioblastoma in combination with standard postoperative chemoradiation therapy, reported that cilengitide did not show improved outcomes, and neither progression-free survival nor overall survival was significantly prolonged [46]. However, the therapeutic approach of targeting integrin remains a sound strategy for high-grade gliomas, and there have been various studies on treatment using RGD [28,46]. In addition, the CENTRIC study reported no additional toxic effects of cilengitide. In the case of high-grade gliomas, higher the malignancy of the glioma, the higher the expression of integrin α v β 3 , which is a poor prognostic factor. Therefore, this novel boron carrier has the potential to be effective for high-grade gliomas. In this study, cRGD was used as a tumor target domain for binding with MID-AC.
Thus, cRGD-MID-AC, which has human serum albumin as a DDS and integrin α v β 3 as a tumor-targeting system, has been developed. The efficacy of human serum albumin and its accumulation in the tumor by conjugation with albumin have been reported in previous studies [16][17][18][19]47,48]. In this experiment, immunogenic HSA was used instead of rat serum albumin. When considering this albumin-containing drug as a pre-clinical study, the results of this study require to be interpreted carefully, taking into account the effects of immunogenicity in a rat brain tumor model. The most significant feature of cRGD-MID-AC is the high retention of cellular boron concentration owing to the tumor accumulation mechanism of albumin as well as MID-AC. In vitro cellular uptake experiments showed that the cellular boron concentration of BPA was highest at all times from 1 h to 24 h. The cellular boron concentration of cRGD-MID-AC increased gradually over time, and the retention rate after 24 h of exposure was much higher than that of BPA ( Figure 1). As BPA is rapidly cleared after exposure, it must be continuously administered intravenously during neutron irradiation in clinical BNCT [3,15]. In contrast, cRGD-MID-AC can retain cellular boron for a long time after the completion of exposure, which is a favorable aspect for BNCT. Furthermore, in assessing the novel boron carrier in BNCT, it is necessary to evaluate the biological effects of BNCT, which are shown by the CBE factor specific for each boron carrier. In this study, based on in vitro neutron-irradiation experiments, the CBE for cRGD-MID-AC was calculated as 2.26 (CBE to be 2.69), which was found to have a sufficient cell-killing effect in BNCT as compared to that of BPA even though the cellular boron concentration of cRGD-MID-AC was lower than that of BPA ( Figure 2).
The efficacy of cRGD-MID-AC as a tumor-targeting system was demonstrated by in vivo biodistribution experiments (Figure 3). In the case of cRGD-MID-AC, the boron concentration in the tumor was as high as that in BPA. Notably, the distribution of boron-10 in normal brain tissue was much lower with cRGD-MID-AC than with BPA, indicating an even lower level of damage from neutron irradiation. Thus, cRGD-MID-AC may provide boron-10 more selectively than BPA, resulting in a safer or more intense boron neutron capture reaction with a longer irradiation time for high-grade gliomas. Furthermore, a previous study showed that BNCT using cRGD-MID-AC is much more effective than BNCT using MID-AC in a U87MG xenograft subcutaneous tumor mouse model because of conjugation with RGD and might enhance therapeutic efficacy against high-grade gliomas with high integrin expression [29]. In addition, compared with the intravenous administration of 20 mg B/kg of MID-AC to the F98 glioma-bearing rat brain tumor model in our previous study, the boron concentration in the tumor was higher when 12 mg/kg of cRGD-MID-AC was administered intravenously [16]. These results suggest that the bonding of cRGD to MID-AC, as a tumor-targeting system, is expected to improve therapeutic efficiency.
In vivo neutron-irradiation experiments showed the efficacy of BNCT using cRGD-MID-AC ( Figure 4A,B). No significant differences were observed between the BNCT with BPA and BNCT with cRGD-MID-AC. Concerning long-term survival in the cRGD-MID-AC 8 h group, which was not observed in the BNCT with BPA group, it is possible that prolonged exposure may enhance cellular boron accumulation in the tumor (even in vitro), and this phenomenon was confirmed in the survival evaluation after BNCT. BPA has been found to be widely distributed in the cytoplasm and cell nucleus. However, the cellular distribution of cRGD-MID-AC remains unclear. Although the interaction between RGD and integrins has long been known, the mechanism of internalization by binding of RGD peptides to integrins has not yet been elucidated. However, it has been reported that at least the endocytosis of integrin α v β 3 is mediated via clathrin-dependent endocytosis or uncoated vesicles [49,50]. Schraa et al. showed that internalization of monomeric RGD ligands is independent of their α v β 3 receptor and occurs via a liquid-phase endocytic pathway. In contrast, multimeric RGD molecules are co-internalized with their receptor, with evidence supporting the aggregation and clustering of integrins [51]. More recently, Sancey et al. reported a peptide-like scaffold with four cRGD motifs, called RAFT-RGD, that target integrin α v β 3 and promote integrin cluster formation. They have further demonstrated that the addition of 1 µmol/L of this molecule (RAFT-RGD) increases the internalization of α v β 3 via clathrin-coated vesicles by 79% [52]. Our study did not examine the internalization rate of integrin α v β 3 or the extent to which our DDS applied with cRGD contributed to the internalization of integrin α v β 3 . However, we will explore these in future studies to better understand and improve the potential of DDS to specifically deliver and retain boron in target cells.
The accumulation of cRGD-MID-AC in glioma cells in the brain partly results from the disruption of the blood-brain barrier (BBB). In BNCT for high-grade gliomas, the requirements for boron-10 carriers include low intrinsic toxicity, high boron accumulation for target lesions, low uptake into normal tissues, water solubility [35,53], and the ability to overcome the BBB and the blood-brain tumor barrier (BBTB) [54][55][56]. Currently, only two boron carriers, namely BPA and sodium borocaptate (BSH), are used in clinical BNCT. BSH contains 12 boron atoms per molecule, reaches tumor cells through disruption of the BBB, and is not cell selective [53]. Because MID, which constitutes cRGD-MID-AC, is a derivative of BSH, cRGD-MID-AC, like BSH, is thought to reach tumor cells by disrupting the BBB. In addition, because integrin α v β 3 is highly expressed in the neovascular vessels surrounding tumors [22,23], cRGD-MID-AC may accumulate more in these neovascular vessels than in normal vessels. If cRGD-MID-AC crosses the BBB, the accumulation of boron in the normal brain is expected to be slightly higher; however, the fact that it is much lower than that of BPA suggests that it does not pass through the healthy, intact BBB. cRGD-MID-AC and MID-AC contain albumin as the DDS, and the accumulation of sufficient boron concentration in tumor cells may result from the albumin conjugate in addition to the MID properties. Therefore, it is very appealing that cRGD-MID-AC with 12 boron atoms per molecule can reach glioma cells and remain in these cells for a long time owing to the properties of human serum albumin.
In another perspective study, cRGD-MID-AC was shown to have potential clinical applications. Recently, in neuro-oncology, positron emission tomography (PET) imaging has attracted much attention because it allows noninvasive assessment of molecular and metabolic processes [57]. In patients with brain tumors, particularly gliomas, it is important to assess treatment-related changes such as radiation necrosis and indications for targeted therapies such as integrins [57]. It has been shown that 18 F-Galacto-RGD PET can assess integrin α v β 3 expression in mouse tumor models and in patients with head and neck cancer. A correlation has also been demonstrated between galacto-RGD uptake in patients with malignant gliomas and the expression of integrin α v β 3 in the corresponding tumor samples [58,59]. These findings suggest that imaging of integrin α v β 3 expression in patients with malignant glioma by 18 F-Galacto-RGD positron emission tomography has already been established [23]. New RGD peptide ligands are also being developed for PET imaging of α v β 3 integrin, which may become even more important as integrin-related therapeutic decisions and treatments become relevant for patients with glioma [60]. Therefore, the application of these techniques would be useful for visually identifying eligible cases for integrin-targeted BNCT. In this study, only rat gliomas and gliosarcoma cells were evaluated. For cRGD-MID-AC to be clinically applicable, it will also be necessary to evaluate other more heterogeneous tumor models in which therapeutic effects can be expected depending on the degree of integrin α v β 3 expression.
Only a few boron-10 carriers can be as effective as or even more effective than BNCT with BPA against high-grade gliomas, especially when administered intravenously [16,34]. This study showed that BNCT with cRGD-MID-AC could precisely target glioma cells even in the brain. This biological targeting of F98 glioma cells that overexpress integrins α v β 3 , apart from LAT-1-targeted BPA, would provide a novel target for BNCT against high-grade gliomas with a heterogeneous nature. Because cRGD-MID-AC has a very low boron distribution in normal brain tissues, it would be expected to further improve the therapeutic effect owing to the different biological targets from to that of BPA.

Conclusions
cRGD-MID-AC, cyclic RGD-functionalized closo-dodecaborate albumin conjugates with maleimide, has been shown to have a therapeutic effect in BNCT in an experimental F98 glioma-bearing rat brain tumor model. The effect of albumin as a DDS increases the blood residence time, and the effect of cRGD as a biological target increases tumor selectivity, which provides more intensive BNCT against high-grade gliomas compared with that with BPA. The targeting strategy of cRGD can achieve a higher tumor boron concentration than the original long-retention type boron carrier, MID-AC, and can improve the therapeutic intensity of BNCT. Furthermore, cRGD-MID-AC is appealing because it can be administered intravenously like BPA. As in the case of MID-AC, it is expected to contribute to the flexible application of neutron irradiation and the performance of BNCT by retaining boron in the tumor for a long period of time. In addition, because of different accumulation mechanisms, it may be possible to use it in combination with BPA-based BNCT to make it a multi-targeted NCT.  Acknowledgments: This work was partly performed under the Research Program for Next Generation Young Scientists of "Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials" in "Network Joint Research Center for Materials and Devices" with funds for K.T. and supervised by H.N. The authors would like to acknowledge Rolf Barth (Ohio State University, Columbus, OH, USA) for providing F98 rat glioma cells. The authors thank Aya Sunamura for her secretarial work and Itsuko Inoue for her technical assistance.