Water-soluble closo-docecaborate-containing pteroyl derivatives targeting folate receptor-positive tumors for boron neutron capture therapy

Article abstract of DOI:10.3390/cells9071615

Water-soluble pteroyl-closo-dodecaborate conjugates (PBCs 1–4), were developed as folate receptor (FRα) targeting boron carriers for boron neutron capture therapy (BNCT). PBCs 1–4 had adequately low cytotoxicity with IC₅₀ values in the range of 1~3 mM toward selected human cancer cells, low enough to use as BNCT boron agents. PBCs 1–3 showed significant cell uptake by FRα positive cells, especially U87MG glioblastoma cells, although the accumulation of PBC 4 was low compared with PBCs 1–3 and L-4-boronophenylalanine (L-BPA). The cellular uptake of PBC 1 and PBC 3 by HeLa cells was arrested by increasing the concentration of folate in the medium, indicating that the major uptake mechanisms of PBC 1–3 are primarily through FRα receptor-mediated endocytosis.

Full text of DOI:10.3390/cells9071615

cells
Article
Water-Soluble closo-Docecaborate-Containing Pteroyl
Derivatives Targeting Folate Receptor-Positive
Tumors for Boron Neutron Capture Therapy
Fumiko Nakagawa 1, Hidehisa Kawashima 2 , Taiki Morita 2 and Hiroyuki Nakamura 1,2,
*
1
School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku,
Yokohama 226-8503, Japan; fu-nakagawa@hitachi-chem.co.jp
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan; kawashima@ims.tsukuba.ac.jp (H.K.);
t.morita@res.titech.ac.jp (T.M.)
2
*
Correspondence: hiro@res.titech.ac.jp; Tel.: +81-45-924-5244
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 17 May 2020; Accepted: 30 June 2020; Published: 3 July 2020
Abstract: Water-soluble pteroyl-closo-dodecaborate conjugates (PBCs 1–4), were developed as folate
receptor (FR
adequately low cytotoxicity with IC50 values in the range of 1~3 mM toward selected human cancer
cells, low enough to use as BNCT boron agents. PBCs 1–3 showed significant cell uptake by FR
α) targeting boron carriers for boron neutron capture therapy (BNCT). PBCs 1–4 had
α
positive cells, especially U87MG glioblastoma cells, although the accumulation of PBC 4 was low
compared with PBCs 1–3 and L-4-boronophenylalanine (L-BPA). The cellular uptake of PBC 1 and PBC
3 by HeLa cells was arrested by increasing the concentration of folate in the medium, indicating that
the major uptake mechanisms of PBC 1–3 are primarily through FRα receptor-mediated endocytosis.
Keywords: boron neutron capture therapy; folate; FRα; closo-dodecaborate; water-soluble
1. Introduction
Boron neutron capture therapy (BNCT) has been attracting attention as a noninvasive radiotherapy
in cancer treatment. Although the energy of the thermal neutron is extremely low (~0.5 eV), the 10
B
neutron capture reaction produces high linear energy alpha particles (4He) and 7Li nuclei that dissipate
their energy (2.4 MeV) while passing through the cell diameter (approximately 5–9 m) in tissue, giving
µ
a fatal cell-killing effect to cancer cells. Therefore, the combination of boron agents with sufficient
and selective accumulation of 10B in cancer cells and an appropriate neutron source is essential for
successful cancer treatment with BNCT [
generators for BNCT have been developed worldwide [
as a medical device [ ] in combination with L-4-boronophenylalanine (L-BPA) [11] for the treatment
of head and neck carcinoma patients. It is known that L-BPA is actively accumulated into cancer cells
through L-type amino acid transporter 1 (LAT-1) [12 13], which is overexpressed in many cancer cells.
1,
2]. In the last decade, accelerator-based thermal neutron
3
–10], and one was approved in Japan this year
6,7
,
However, there are still many patients for whom L-BPA is not applicable. One of the reasons for low
L-BPA accumulation in some patients’ tumors may be the low expression of LAT-1 in their tumors;
thus the development of novel boron carriers applicable to various cancers including L-BPA-negative
tumors is required for further development of BNCT.
Folate is one of the B vitamins and is necessary for the synthesis of purines and thymidine
as well as for methylation of DNA, proteins and lipids via S-adenosyl methionine [14]; thus it
is especially important for frequent cell division [15]. Folate is known to be taken into cells via
the folate receptors, cysteine-rich cell-surface glycoproteins [16]. Since the folate receptors are
overexpressed on the surface of many cancer cells including HeLa and U-87 MG [17,18], they have
Cells 2020, 9, 1615; doi:10.3390/cells9071615
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attracted attention as targets for cancer treatment [19
–21]. Various folate receptor-targeted therapies for
cancer have been investigated including folate-toxin conjugates, folate-conjugated chemotherapeutic
agents, folate receptor-targeted immunotherapy, and folate-conjugated liposomes, micelles, and other
nanoparticles [22]. The folate receptor-targeted approach has also been used to develop boron delivery
vehicles, such as liposomes [23], polyamidoamine dendrimers [24], boron nanoparticles [25] and
nanotubes [26
molecular-weight boron agents [29
,
27], and gold nanoparticles [28], in BNCT. However, there are few reports on low
30]. In all cases, the lipophilic ortho-carborane, as the source of
,
boron, was conjugated with folate, so the boron carriers did not possess adequate water solubility for
administration. In fact, 500 mg/kg L-BPA is clinically administrated to achieve the required boron
concentration in the tumor [31]. Thus, the water solubility of boron carriers is one of the essential
requirements. We focused on a closo-dodecaborate, a water soluble and low toxicity boron cluster,
and introduced it into folate. It is known that the pteroyl group of folate is essential for the interaction
with folate receptors [32,33]. In this paper, we synthesized pteroyl-closo-dodecaborate conjugates
(PBCs) and examined their cell uptake using folate receptor (FRα) positive and negative cells.
2. Materials and Methods
2.1. General Methods
Boron concentrations were measured with inductively coupled plasma optical emission
spectroscopy (ICP-OES) (Thermo Fisher Scientific Inc. Waltham, MA, USA). The statistical significance
of the results was analyzed using the Student’s t-test for unpaired observations and Dunnett’s test for
multiple comparisons. All protocols for in vivo study were approved by the Institutional Animal Care
and Use Committee of Tokyo Institute of Technology (D2015011). (Et3NH)2[B12H12] was purchased
from Katchem Ltd. (Prague, Czech Republic), and Na2[10B12H12] was kindly provided by Stella
Chemifa Co. (Osaka, Japan). Other chemicals were purchased from Tokyo Chemical Industry Co., Ltd.
(TCI). HeLa (human cervix epithelioid carcinoma) and A549 (human alveolar adenocarcinoma) cells
were kindly provided by the Cell Resource Center for Biomedical Research, Institute of Development,
Aging and Cancer, Tohoku University. U-87 MG (human malignant glioma) cells were purchased from
Cosmo Bio Co., Ltd.
2.2. MTT Assay
HeLa, U-87 MG, and A549 were seeded in 96-well plates in RPMI-1640 medium (Wako Pure
Chemicals) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco; Thermo
Fisher Scientific, Waltham, MA, USA) at the density of 5
×
103 cells/well. After 24 h of cell attachment,
the cells were exposed to PBC 1, PBC 2, PBC 3, PBC 4, and L-BPA-fructose complex at final concentrations
ranging from 0.1 to 10 mM for 72 h at 37 ◦C. At the end of the incubation period, the mitochondrial
function was verified with 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium
bromide) for 2 h at 37 ◦C and quantified spectrophotometrically at 595 nm by Biorad microplate reader.
Data were expressed as a percentage of the viability of each control.
2.3. Cell Uptake Study
PBCs or L-BPA solutions were prepared from PBCs (1500–3000 ppm B dissolved in ultrapure
water (Milli-Q water)) or L-BPA (2400 ppm B in the fructose solution) and medium. HeLa, U-87 MG,
and A549 cells were cultured at the density of 3
×
105 cells/dish (U-87 MG) or 1
×
106 cells/dish (other
cells) in 6-well plates in the medium (1mL) at 37 ◦C in a 5% CO2 incubator for 24 h; then the medium
was removed and replaced with the drug solutions for 1, 3, and 12 h. This medium was removed
and the cells were washed three times with PBS (phosphate-buffered saline), collected by rubber,
and dissolved in a mixed solvent of 60% HClO4 and 30% H2O2 (1:2 v/v) at 70 ◦C for 2 h, and then Milli-Q
water was added up to 5 mL total. After filtering through a membrane filter (0.5
µmϕ, 13JP050AN,
ADVANTEC, Japan), the boron concentration of the resulting solutions was measured by ICP-OES.

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2.4. Immunofluorescence
HeLa cells were plated into
ϕ
35 dishes (1
×
104 cells) including the glass coverslips (10 mm
◦
square) and incubated at 37 C for 24 h. After PBC 1 (100 ppm B) treatment for 3 h, the cells
were washed three times with PBS and fixed with 4% paraformaldehyde in PBS for 10 min.
After washing with 0.4 % Triton X-100 in PBS for 5 min, the cells were incubated with anti-BSH
(mercaptoundecahydro-closo-dodecaborate) antibody at 4 ◦C overnight. After washing three times with
PBS, the cells were incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody
for 2 h, the cells were washed three times with PBS, and tyramide-Cy3 was added. After incubating for
5 min, the cells were washed three times with PBS, and DAPI (4’,6-diamidino-2-phenylindole) solution
was added. After incubating for 5 min, the cells were washed three times with PBS and mounted with
Prolong Gold anti-fade reagent (invitrogen). Fluorescence images were analyzed using a confocal laser
scanning microscope (Carl Zeiss, LSM780).
3. Results
3.1. Design and Synthesis of Pteroyl Closo-Dodecaborates
Although the synthesis of differentially functionalized folate derivatives is not an easy task, Fuchs
et al., have developed effective protocols through pteroyl azide [34]. Thus, we designed PBCs 1–4 from
pteroyl azide and closo-dodecaborate 1 [35] conjugated with amino acid linkers (Figure 1).
O
2-
O
Linker
O
O
N
N
H
N
N
= BH
= B
HN
H2N
N
H
closo-dodecaborate
(h y d ro p h ilic )
Pteroyl closo-dodecaborates
(PBCs)
O
-
H3N
O
O
N
N3
O
Amino acid
+
+
N
N
HN
H2N
N
linker
CO2H
H2N
H
1
Pteroyl azide
Compound
PBC1
Linker
CO2H
H
N
PBC2
PBC3
PBC4
(glutamine)
(glycine)
O
H
N
O
CH3
H
N
(alanine)
O
Figure 1. Structures and synthetic strategy of water-soluble pteroyl closo-dodecaborates (PBCs1-4).
Synthesis of amino acid linker-conjugated closo-dodecaborates is shown in Scheme 1.
According to the reported procedure with modification [36], closo-dodecaborate
synthesized from bis-tetrabutylammonium form , which was easily prepared from the
commercially available closo-dodecaborate bis-sodium form, through the dioxane complex
Then closo-dodecaborate was treated with 1-benzyl-N-tert-butoxycarbonyl-L-glutamate using
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), N,N-dimethylaminopyridine (DMAP),
and triethylamine (TEA) to give compound in 68% yield. Hydrogenesis to remove the benzyl
protective group followed by acid hydrolysis afforded compound in 71% yield. Similarly,
glycine and alanine linked closo-dodecaborates and were prepared from compound with
1 was
2
3.
1
4
5
6
8
1
N-tert-butoxycarbonyl-L-glycine and N-tert-butoxycarbonyl-L-alanine, respectively, and the acid
hydrolysis to remove the Boc protection gave compounds 7 and 9, quantitatively.

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Scheme 1. Synthesis of closo-dodecaborate moieties. Reaction conditions: (a) 1,4-dioxane, NaBF4,
HCl, 100 ◦C, 18 h, 78%; (b) i. 25% NH3 aq. CH3CN, 50 ◦C, ii. 10% TBAOH, MeOH;
(c) 1-benzyl-N-tert-butoxycarbonyl-L-glutamate, EDCI, DMAP, TEA, CH2Cl2, r.t., 68%; (d) i. H2,
Pd/C, MeOH, r.t., ii. 4N HCl, 1,4-dioxane, 71%; (e) N-tert-butoxycarbonyl-L-glycine, EDCI, DMAP, TEA,
CH2Cl2, r.t., 94%; (f) 4N HCl, 1,4-dioxane, 84%~quant.; (g) N-tert-butoxycarbonyl-L-alanine, EDCI,
DMAP, TEA, CH2Cl2, r.t., 85%. TBA = tetrabutylammonium. Bn = benzyl. Boc = tert-butoxycarbonyl.
PBCs 1–4 were synthesized as shown in Scheme 2. First, pteroyl azide was synthesized
from folate through compound 10 according to the reported procedures [31] and conjugated with
closo-dodecaborates 1, 5, 7, and 9 in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
The resulting TBA forms of pteroyl derivatives were converted to bis-tetramethylammonium forms
followed by bis-sodium forms to afford PBCs 1–4. The chemical structures of PBCs 1–4 were identified
by 1H, 13C, and 11B NMR, high resolution mass spectroscopy, and IR (see the supporting information).
O
CO2H
O
O
CO2H
O
N
N
CO2H
O
N
N
O
N
N3
a
b
c
H
N
N
PBC 1-4
N
N
N
N
HN
H2N
N
HN
H2N
N
H
HN
H2N
N
O
H
H
Folate
10
Pteroyl Azide
Scheme 2. Synthesis of PBCs 1–4. Reaction conditions: (a) i. trifluoroacetic anhydride, THF, 0 ◦C,
ii. H2O, THF, r.t.; (b) i. N2H4 H2O, DMSO, r.t., ii. NaN3, KSCN, t-BuONO, trifluoroacetic acid; (c) i.
closo-dodecaborates , or
, DBU, DMSO, r.t., ii. TMACl, MeOH, r.t., iii. Amberlite Na+ form, H2O,
·
1,
5,
7
9
r.t. TMA = tetramethyl ammonium.
3.2. Cytotoxicity of PBCs
As mentioned earlier, a high dose is necessary to achieve the required boron concentration in the
tumor for BNCT. Thus, not only adequate water solubility but also low cytotoxicity is essential for
BNCT boron agents. We first examined the cytotoxicity of synthesized PBCs toward three human
cancer cell lines using MTT assay: HeLa (human cervical carcinoma) and U87MG (human glioblastoma)
cells are FRα positive and A549 (human alveolar adenocarcinoma) cells are FRα negative (see Figure
S1 in the supporting information). L-BPA was used as a positive control. The results are summarized
in Table 1. PBCs 1–4 exhibited IC50 values (the concentrations required for 50% inhibition) in a range
of 1–3 mM toward these human cancer cells, indicating that PBCs 1–4 have adequately low cytotoxity,
enough to use as BNCT boron agents.

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Table 1. Cytotoxicity of PBCs 1–4 toward HeLa, U87MG, and A549 cells a.
IC50 (mM) b
Compound
HeLa
U87MG
A549
PBC 1
PBC 2
PBC 3
PBC 4
L-BPA
2.00 ± 0.31
2.04 ± 0.05
2.53 ± 0.54
1.51 ± 0.47
>10
1.79 ± 0.46
2.29 ± 0.37
1.67 ± 0.16
1.57 ± 0.72
>10
3.03 ± 0.12
1.86 ± 0.11
1.15 ± 0.15
2.08 ± 0.24
>10
a
Cells were incubated for 72 h with various concentrations (10
µ
M–10 mM) of compounds, and cell viability
b
was determined by the MTT assay. The drug concentration required to inhibit cell growth by 50% (IC50) was
determined from semi-logarithmic dose–response plots, and results represent means ± SD of triplicate samples.
3.3. Cellular Uptake and Distribution of PBCs
We next examined the boron accumulation of PBCs 1–4 in these three cell lines. The results are
shown in Figure 2. Both PBC 1 and PBC 2 were similarly accumulated into HeLa cells, whereas twice
the boron accumulation was observed in the case of PBC 3 and L-BPA. To our surprise, the cell uptake
of PBC 4 was quite low compared with PBCs 1–3. A similar tendency was observed in the accumulation
into U87MG cells. Interestingly, the boron accumulation of PBCs 1–3 was higher than that of L-BPA.
It is known that L-BPA is accumulated into cells through LAT1 and that the expression level of LAT1
is relatively low on the surface of U87MG cells. Therefore, the significant accumulation difference
between PBCs 1–3 and L-BPA is probably due to the FRα positive and LAT1 negative property of
U87MG cells. In fact, a lower accumulation of PBCs into FRα negative A549 cells was observed
compared with those of FRα positive HeLa and U87MG cells.
Figure 2. Boron accumulation of PBCs 1–4 into HeLa, U87MG, and A549 cells. The cells (1
×
106 cells)
were incubated at 37 ◦C for 3 h in a medium containing boron compounds (100 ppm B). After incubation,
the cells were washed three times with PBS and digested with 2 mL of perchloric acid/hydrogen
peroxide at 70 ◦C for 1 h. Boron concentrations in the solution were determined by an inductively
coupled plasma optical emission spectroscopy (ICP-OES).
It should be noted that PBC 3 always exhibited significant accumulation into the cells. Therefore,
we carried out folate concentration-dependent boron accumulation of PBC 1 and PBC 3 into HeLa cells
to confirm whether PBCs were accumulated through FRα or not. The results are shown in Figure 3A.
Folate concentration-dependent decreases of boron concentration in HeLa cells were observed in
the cases of PBC 1 and PBC 3; however, the boron accumulation of L-BPA was not affected by the

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presence or absence of folate. We also examined the cellular distribution of PBC 1 in HeLa cells by
immunostaining using anti-closo-dodecaborate antibody. As shown in Figure 3B, PBC 1 was located in
the cytosol where FRα normally resides. These results indicate that the major uptake mechanism of
both PBC 1 and PBC 3 is primarily through FRα receptor-mediated endocytosis.
(A)
*
*
**
**
folate (mM)
(B)
control
PBC1
Figure 3. (A) Folate concentration-dependent boron accumulation of PBC 1, PBC 3, and L-BPA into
HeLa cells. HeLa cells were incubated with PBC 1, PBC 3 (100 ppm B), or L-BPA (25 ppm B) in the
various concentrations of folate. Statistical significance: *p < 0.005 and **p < 0.001 compared with
controls (no folate). (B) Cellular distribution of PBC 1 in HeLa cells. Immunostaining was carried out
using anti-closo-dodecaborate antibody. Red color: the localization of PBC 1; blue color: DAPI-stained
nucleus. Scale bar: 20 µm.
4. Discussion
For targeting FR
α
, the lipophilic ortho-carborane-conjugated folate derivatives have been reported
so far, as boron carriers of BNCT. However, these boron carriers did not possess adequate water
solubility for administration due to the highly lipophilic property of ortho-carborane. In the present
study, we have prepared pteroyl-closo-dodecaborate conjugates, PBCs 1–4 as FRα targeting boron
carriers. By using closo-dodecaborate, instead of lipophilic ortho-carborane, PBCs 1–4 showed adequate
water-solubility as expected. Combined with low cytotoxicity of PBCs 1–4 which was indicated by
MTT assay, these compounds seem to have appropriate properties as BNCT boron agents. In fact,
as shown in Figure 2, the cellular uptake study demonstrated significant accumulation of PBCs 1–3
into FR
α
positive cells, though PBC-4 showed much lower accumulation than PBCs 1–3. The difference
-position of the amide bond in PBCs.
between PBC 3 and PBC 4 is a methyl group substituted at the
α
These results suggest that the structure of the amino acid linker is highly important for the property of
PBCs. Although the studies on the folate concentration-dependent boron accumulation demonstrated
that PBC 1 and PBC 3 were accumulated through FRα receptor-mediated endocytosis, PBC 3 still
showed high accumulation into FR negative A549 cells. This result would indicate that PBC 3 tends
α
to accumulate into cells because of their relatively lipophilic glycine linker. Indeed, as we observed
the precipitation of PBC 3 during the biodistribution study (date not shown), even a slight structural

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modification would have a drastic impact. We believe that further investigation on the structure of
the linker moiety will lead to sufficient boron accumulation for BNCT treatment. It should be noted
that folate concentration-dependent decreases of boron concentration in HeLa cells were observed
in the cases of PBC 1 and PBC 3 as shown in Figure 3A. However, the boron accumulation of L-BPA
was not affected by the presence or absence of folate. These results indicate that both PBCs and L-BPA
have different uptake pathways. It is known that L-BPA accumulates into tumor cells primarily via the
LAT-1 pathway; thus, simultaneous administration of both PBCs and L-BPA can enhance boron uptake
by tumor cells. In fact, the survival time of brain tumor model rats treated with both PBC 1 and L-BPA
was significantly prolonged in comparison with those treated with PBC 1 or L-BPA alone. [37]
5. Conclusions
We designed and synthesized water-soluble pteroyl-closo-dodecaborate conjugates, PBCs 1–4, as
FR
of 1~3 mM toward these human cancer cells, low enough to use as BNCT boron agents. PBCs 1–3
showed significant cell uptake by FR positive cells, especially U87MG glioblastoma cells, although
α targeting boron carriers. PBCs 1–4 have adequately low cytotoxicity with IC50 values in the range
α
the accumulation of PBC 4 was low compared with PBCs 1–3 and L-BPA. The cellular uptake of PBC
1 and PBC 3 by HeLa cells was arrested by increasing the concentration of folate in the medium,
indicating that the major uptake mechanisms of PBCs 1–3 are primarily through FRα receptor-mediated
endocytosis. Therefore, newly synthesized PBCs 1–3 have the potential to be alternative boron agents
that could be applied to various cancers including L-BPA-negative tumors for the further development
of BNCT.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/7/1615/s1,
Figure S1: Western blot analysis of FR
folate receptor (FR
α in various cell lines. Figure S2: Quantitative analysis of
α
) by flow cytometry. Figure S3: Time-dependent boron accumulation in HeLa cells.
Figure S4: The concentration-dependent cell viability of folate in HeLa cells.
Author Contributions: Conceptualization, H.K. and H.N.; methodology, F.N., H.K., T.M.; investigation, F.N.,
H.K.; data curation, F.N.; writing—original draft preparation, H.N.; writing—review and editing, T.M., H.N.;
supervision, H.N.; funding acquisition, H.N. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was partially supported by Fundamental Cancer Research (19cm0106262h0001) from the
Japan Agency for Medical Research and Development (AMED).
Acknowledgments: We are grateful to Open Research Facilities for Life Science and Technology, Tokyo Institute
of Technology for support of the analysis of fluorescence images.
Conflicts of Interest: The authors declare no conflict of interest.
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