Folate-appended β-cyclodextrin as a promising tumor targeting carrier for antitumor drugs in vitro and in vivo

Article abstract of DOI:10.1021/bc400015r

A large number of antitumor drug delivery carriers based on passive targeting and/or active targeting have been developed. However, encapsulation of antitumor drugs into these drug carriers is often complicated, and antitumor activities of these targeting systems are not satisfactory. In the present study, we first prepared heptakis-6-folic acid (FA)-appended β-cyclodextrin (β-CyD) possessing two caproic acids between FA and a β-CyD molecule as a spacer (Fol-c₂-β-CyD) and evaluated the potential as a novel tumor targeting carrier for antitumor drugs through a complexation. Fol-c₂-β-CyD formed an inclusion complex with doxorubicin (DOX) at a 1:1 molar ratio with a markedly high stability constant (>10⁶ M^-1). Cellular uptake of DOX was increased by the addition of Fol-c₂-β-CyD in KB cells, a folate receptor-α (FR-α)-positive cell line. Additionally, Fol-c₂-β-CyD increased in vitro antitumor activities of antitumor drugs such as DOX, vinblastine (VBL), and paclitaxel (PTX) in KB cells, but not in A549 cells, a FR-α-negative cell line. The complex of DOX with Fol-c₂-β- CyD markedly increased antitumor activity of DOX, not only after intratumoral administration but also after intravenous administration to mice subcutaneously inoculated Colon-26 cells, a FR-α-positive cell line. These findings suggest that Fol-c₂-β-CyD could be useful as a promising antitumor drug carrier.

Full text of DOI:10.1021/bc400015r

Article
pubs.acs.org/bc
Folate-Appended β‑Cyclodextrin as a Promising Tumor Targeting
Carrier for Antitumor Drugs in Vitro and in Vivo
Ayaka Okamatsu,† Keiichi Motoyama,† Risako Onodera,† Taishi Higashi,† Takahiro Koshigoe,‡
Yasutaka Shimada,§ Kenjiro Hattori,‡,§ Tomoko Takeuchi,‡ and Hidetoshi Arima*,†
†Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
‡Faculty of Engineering, Tokyo Polytechnic University, 1583 Iiyama, Atsugi 243-0297, Japan
§R&D Lab, NanoDex Inc., 705-1 Shimoimaizumi, Ebina 243-0435, Japan
S
* Supporting Information
ABSTRACT: A large number of antitumor drug delivery carriers based on passive targeting and/or active targeting have been
developed. However, encapsulation of antitumor drugs into these drug carriers is often complicated, and antitumor activities of
these targeting systems are not satisfactory. In the present study, we first prepared heptakis-6-folic acid (FA)-appended β-
cyclodextrin (β-CyD) possessing two caproic acids between FA and a β-CyD molecule as a spacer (Fol-c2-β-CyD) and evaluated
the potential as a novel tumor targeting carrier for antitumor drugs through a complexation. Fol-c2-β-CyD formed an inclusion
complex with doxorubicin (DOX) at a 1:1 molar ratio with a markedly high stability constant (>106 M−1). Cellular uptake of
DOX was increased by the addition of Fol-c2-β-CyD in KB cells, a folate receptor-α (FR-α)-positive cell line. Additionally, Fol-c2-
β-CyD increased in vitro antitumor activities of antitumor drugs such as DOX, vinblastine (VBL), and paclitaxel (PTX) in KB
cells, but not in A549 cells, a FR-α-negative cell line. The complex of DOX with Fol-c2-β-CyD markedly increased antitumor
activity of DOX, not only after intratumoral administration but also after intravenous administration to mice subcutaneously
inoculated Colon-26 cells, a FR-α-positive cell line. These findings suggest that Fol-c2-β-CyD could be useful as a promising
antitumor drug carrier.
active targeting ability to tumor tissues and cells is strongly
expected to enhance the therapeutic effects and to reduce any
adverse effects.
INTRODUCTION
■
Although many antitumor drugs are used in the pharmaceutical
field, they have significant adverse effects because of nonspecific
delivery and/or drug resistance in cancer cells. Drug delivery
system (DDS) such as targeting is one of the most promising
approaches to improve adverse effects of antitumor drugs.
Targeting techniques are classified into passive targeting and
active targeting, and the former is based on the enhanced
permeability and retention (EPR) effect.1−3 The representative
product possessing passive targeting ability is Doxil, which is
polyethylene glycol (PEG)-modified liposome containing
doxorubicin (DOX). Doxil can avoid capture from the
reticuloendothelial system (RES) because of the introduction
of PEG with liposomes.4−6 Although passive targeting is useful
in cancer chemotherapy, it is difficult to completely eliminate
the adverse effects of antitumor drugs.4 Therefore, the
development of drug carriers possessing both passive and
To achieve active targeting drug delivery, a chemical
modification of drug carriers with tumor targeting ligands
such as sugar,7 transferrin,8,9 antibody,10 peptides,6,11,12 and
folic acid (FA)13,14 is utilized. Of these ligands, FA is one of the
most widely used because of the following advantages:15,16 (1)
folate receptor-α (FR-α) is highly expressed in many human
tumor cells, including malignancies of the ovary, kidney, breast,
myeloid cells, brain, and lung, (2) FA can be recognized by FR-
α with high binding constants (Kd ∼ 1010 M−1), (3) high
compatibility with a variety of organic and aqueous solvent, (4)
low immunogenicity, (5) low molecular weight (MW 441.4),
Received: January 8, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/bc400015r | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
and (6) low cost. Hence, a large number of folate-appended
drug carriers, e.g., liposomes,13,17−19 dendrimers,20 polysac-
charides,21,22 and micelles,23 have been developed. However,
encapsulation of antitumor drugs into these drug carriers is
often complicated, and antitumor activities of these targeting
systems are not satisfactory. Recently, folate-conjugated vinca
alkaloids, EC145, have emerged as a candidate for clinical
development.24 In this conjugate, however, only one FA was
modified in one drug molecule, highly possibly resulting in
lower binding to FR-α than FA. Thus, folate-appended drug
carriers which enable easiest drug encapsulation and more
effective drug delivery are required.
Cyclodextrins (CyDs) were first isolated in 1891 as
degradation products of starch, and α-, β-, and γ-CyDs are
the most common natural CyDs, consisting of six, seven, and
eight glucose units, respectively. CyDs are cyclic oligosacchar-
ides forming inclusion complexes with a wide range of
hydrophobic molecules, and are utilized as drug delivery
carriers.25−29
Recently, some folate-appending CyDs as tumor-targeting
drug delivery carriers have been reported. Salmaso et al.30,31
demonstrated that the conjugate of FA with β-CyD through a
PEG spacer promotes the uptake of rhodamine-B into KB cells,
FR-α-overexpressing human epidermal carcinoma, but not into
MCF7 cells, non-FR-α expressing human lung carcinoma cell
line. Also, Zhang et al.32 prepared the FA-modified β-CyD with
a PEG spacer through a click chemistry strategy. This conjugate
forms nanoparticle by entrapping 5-fluorouracil (5-FU) in
aqueous solution, and the resulting nanoparticle shows selective
uptake through FR-α-mediated endocytosis in the FR-α-
overexpressing cells. However, there are few studies which
achieved antitumor drug delivery in vivo using FA-conjugated
CyDs. Generally, inclusion complexes of drugs with CyDs
rapidly dissociate to free CyDs and free drugs after parenteral
administration due to their weak interaction.33 Besides,
multivalent interaction between carriers and targeting receptors
promotes the recognition of ligands by the receptors.34,35 Thus,
to achieve in vivo antitumor drug delivery using FA-conjugated
CyDs, the optimal carrier design, which allows strong
interaction between CyDs and drugs, as well as multivalent
interaction between carriers and FR-α, is considered. Hattori et
al.36−40 prepared heptakis-sugar-branched CyDs with spacers
between sugars and CyDs. The wide-open sugar antenna of
these conjugates attracts and interns the drugs in aqueous
solution (sea anemone effect), and these complexes show
markedly high stability constants (Kc ∼ 109 M−1). Moreover,
these conjugates also bind strongly with rat liver cells due to
multivalent interaction between the sugar moieties of
conjugates and the asialoglycoprotein receptor expressing on
hepatocytes (∼1010 M−1).
Nichirei (Tokyo, Japan), respectively. Tetramethyl rhodamine
isothiocyanate (TRITC) was obtained from Funakoshi (Tokyo,
Japan). DOX hydrochloride was donated by Mercian (Tokyo,
Japan). All other chemicals and solvents were of analytical
reagent grade, and deionized double-distilled water was used
throughout the study.
Synthesis of heptakis-6-Cl-β-CyD (Cl-β-CyD, 1). β-CyD
(3.00 g) dehydrated with ethanol was dissolved in dry DMF
(21 mL) containing CaH2 and methylsulfonyl chloride (2.9
mL) under a stream of nitrogen gas, and then the solution was
stirred for 4.5 h at 75 °C. After cooling the solution for 30 min,
ethanol (5 mL) was added and stirred for 30 min to stop the
reaction. Next, 10 mL of sodium methanolate (28% methanol
solution) was added to the reactant, and concentrated using a
rotary evaporator (EYELA N-1000S, Tokyo Rikakikai, Tokyo,
Japan). The sample was filtered and washed with methanol and
H2O. After drying the precipitate under reduced pressure for 16
h at 40 °C, Cl-β-CyD was obtained. The reaction was
monitored by TLC (silica gel F254, Merck, Whitehouse Station,
NJ). Eluent: 1-buthanol/ethanol/water = 5:4:3 (v/v/v).
Indicator: p-anisaldehyde. MALDI-TOF MS [M+H]+ m/z
1266.
Synthesis of heptakis-6-N3-β-CyD (N3-β-CyD, 2). Cl-β-
CyD (1.26 g) was dissolved in dimethylacetamide (DMAc)/
H2O (60:8 v/v), and sodium azide (1.42 g) was added to the
solution. The mixture was stirred for 24 h at 110 °C. After
cooling the solution for 1 h, large volume of H2O was added to
obtain precipitate. After filtration, the sample was dried under
reduced pressure for 13 h at 40 °C, and N3-β-CyD was
obtained. The reaction was monitored by TLC (silica gel F254
,
Merck, Whitehouse Station, NJ). Eluent: 1-buthanol/ethanol/
water = 5:4:3 (v/v/v). Indicator: p-anisaldehyde. MALDI-TOF
MS [M+H]+ m/z 1308.
Synthesis of heptakis-6-NH2-β-CyD (NH2-β-CyD, 3). N3-
β-CyD (1.00 g) and triphenylphosphine (3.20 g) were
dissolved in DMAc (73 mL) and stirred for 1 h at room
temperature. Aqueous ammonia (25%, 73 mL) was added in
the mixture, and stirred for 22.5 h at room temperature. The
reactant was precipitated with H2O (730 mL) and dissolved in
DMAc. And then, the reactant was precipitated and washed
with acetone. After drying under reduced pressure for 20 h at
40 °C, NH2-β-CyD was obtained. The reaction was monitored
by TLC (silica gel F254, Merck, Whitehouse Station, NJ).
Eluent: 1-buthanol/ethanol/water = 5:4:3 (v/v/v). Indicator:
p-anisaldehyde and ninhydrin. MALDI-TOF MS [M+H]+ m/z
1129.
Synthesis of Boc-cap1-OH. 6-Aminohexanoic acid (65.60
g) was dissolved in dioxane/H2O (500:250 v/v), and
triethylamine (84 mL) and di-tert-butyl dicarbonate (131.00
g) were added under stirring for 30 min on ice. Then, the
mixture was stirred for 36 h at room temperature. The reactant
was concentrated using a rotary evaporator, and sodium
hydrogen carbonate (84.02 g) and H2O (500 mL) were
added to the reactant. After adjusting pH at 8.0, the sample was
transferred into separatory funnel, and then washed six times
with acetic ether (200 mL). Next, citric acid (150 g) was added
to the sample and the pH of the sample was adjusted at 3.9.
The sample was extracted four times with acetic ether (200
mL). Sodium sulfate anhydrate (200 g) was added to the
sample, and stirred for 11 h at room temperature. After
filtration, the sample was washed with acetic ether. n-Hexane
(50 mL) was added to the sample, and concentrated by a rotary
evaporator. This operation was repeated ten times. Acetic ether
On the basis of these backgrounds, we first prepared heptakis-
6-FA-appended β-CyD possessing two caproic acids as a spacer
between FA and β-CyD molecules (Fol-c2-β-CyD) in this
study. Then, the potential of this conjugate as an antitumor
drug carrier was evaluated in vitro and in vivo.
EXPERIMENTAL PROCEDURES
■
Materials. β-CyD was donated by Nihon Shokuhin Kako
(Tokyo, Japan). FA was purchased from Nacalai Tesque
(Kyoto, Japan). RPMI-1640 culture medium (FA-free) was
purchased from GIBCO (Tokyo, Japan). Dulbecco’s modified
Eagle’s medium (DMEM) and fetal bovine serum (FBS) were
purchased from Nissui Pharmaceuticals (Tokyo, Japan) and
B
dx.doi.org/10.1021/bc400015r | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
(50 mL) and petroleum ether (30 mL) were added, and
precipitates were extracted by standing the solution for 12 h in
the freezer. Finally, the sample was dried under reduced
pressure for 73 h at room temperature and Boc-cap1-OH was
at 35 °C. The reactant was precipitated with acetone, and
washed five times with acetone (5 mL). Finally, the sample was
purified by Bio-Gel P4 (column, ϕ 4 cm × 69 cm; flow rate,
0.21 mL/min; sample size, 301.3 mg/3 mL 1 M NH3 aq.), and
then Fol-c2-β-CyD was obtained. The reaction was monitored
by TLC (silica gel F254, Merck, Whitehouse Station, NJ).
Eluent: 1-buthanol/ethanol/water/25% NH3 aq = 5:4:3:5 (v/
v/v/v). Indicator: p-anisaldehyde and ninhydrin. MALDI-TOF
MS [M+H]+ m/z 5674.
obtained. The reaction was monitored by TLC (silica gel F254
,
Merck, Whitehouse Station, NJ). Eluent: 1-buthanol/ethanol/
water = 5:4:3 (v/v/v). Indicator: ninhydrin. MALDI-TOF MS
[M+Na]+ m/z 254.
Synthesis of 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium Chloride (DMT-MM). N-Methylmor-
pholine (NMM, 14.8 mL) and 2-chloro-2,6-dimethoxy-1,3,5-
triazin (25.29 g) were dissolved in tetrahydrofuran (THF, 450
mL), and the mixture was vigorously stirred for 1 h at room
temperature. The white precipitate was washed three times with
THF (100 mL). After drying under reduced pressure, DMT-
MM was obtained. MALDI-TOF MS [M-Cl]+ m/z 241.
Synthesis of heptakis-6-Boc-cap1-β-CyD (Boc-c1-β-
CyD, 4). Boc-cap1-OH (6.52 g), NH2-β-CyD (4.32 g) and
DMT-MM (8.48 g) were dissolved in methanol/H2O (35:35
v/v), and stirring for 42 h at room temperature. The reactant
was precipitated by H2O (1200 mL). After filtration, the sample
was washed with H2O and dissolved in methanol. After
condensation and lyophilization, Boc-c1-β-CyD was obtained.
The reaction was monitored by TLC (silica gel F254, Merck,
Whitehouse Station, NJ). Eluent: 1-buthanol/ethanol/water =
5:4:3 (v/v/v). Indicator: p-anisaldehyde and ninhydrin.
MALDI-TOF MS [M+Na]+ m/z 2644.
Synthesis of heptakis-6-NH2-cap1-β-CyD (NH2-c1-β-
CyD, 5). Boc-c1-β-CyD (8.03 g) was dissolved in 4 M HCl/
dioxane (36 mL) and the mixture was stirred for 2 h on ice.
After condensation, the reactant was lyophilized, and then
NH2-c1-β-CyD was obtained. The reaction was monitored by
TLC (silica gel F254, Merck). Eluent: 1-buthanol/ethanol/water
= 5:4:3 (v/v/v). Indicator: p-anisaldehyde and ninhydrin.
MALDI-TOF MS [M+H]+ m/z 1920.
Synthesis of heptakis-6-Boc-cap2-β-CyD (Boc-c2-β-
CyD, 6). NH2-c1-β-CyD (9.65 g) and Boc-cap1-OH (24.30 g)
were dissolved in methanol (210 mL), and NMM (11.1 mL)
and DMT-MM (29.01 g) were added into the mixture. After
agitation for 37 h at room temperature, the reactant was
precipitated with H2O (2 L). The crude product was filtered
and washed five times with H2O (100 mL). The reactant was
dissolved in methanol and concentrated using a rotary
evaporator. The sample was dried under reduced pressure for
73 h at 40 °C, and then Boc-c2-β-CyD was obtained. The
reaction was monitored by TLC (silica gel F254, Merck,
Whitehouse Station, NJ). Eluent: 1-buthanol/ethanol/water =
5:4:3 (v/v/v). Indicator: p-anisaldehyde and ninhydrin.
MALDI-TOF MS [M+Na]+ m/z 3435.
1H NMR Spectrometry. 1H NMR spectra were taken at 25
°C on a JEOL JNM-ECP500 (Tokyo, Japan), operating at 500
MHz, using a 5 mm sample tube. Deuterated water (D2O) and
DMSO (DMSO-d6) were used as solvent. The samples were
dissolved in D2O or DMSO-d6 at a concentration of 5 mg/0.6
mL. The signal of D2O or DMSO-d6 was used as an internal
1
reference for H NMR.
Surface Plasmon Resonance (SPR). The molecular
interaction of DOX with Fol-c2-β-CyD was examined using
an optical biosensor “IAsys” based on SPR (Affinity Sensor,
Cambridge, UK). The immobilization of DOX on the sensor
cuvette was carried out by the reaction of a reactive linker
molecule with the cuvette surface. After activation by washing
with 8 M urea solution containing 10 mM MnCl2, the
interaction curves were measured at the concentration of Fol-
c2-β-CyD (0 to 1 μM) in 10 mM acetate buffer (pH 7.3) with 1
mM CaCl2 and 100 mM NaCl at 25 °C. The association
constant was obtained by measuring the change in the refractive
index according to the usual procedure. The computational
results were derived using a software FAST-fit equipped in the
IAsys.
Fluorescence Spectrometry. DOX was dissolved in
phosphate buffer (pH 7.3 or 6.8) in the presence of various
concentrations of β-CyD, FA-appended β-CyD without a
spacer (degree of substitution of FA, 5; FA-β-CyD), and Fol-c2-
β-CyD. Fluorescence spectra of the samples were measured at
25 °C using F-4500 fluorescence spectrometers (Hitachi,
Tokyo, Japan).
Stoichiometry. The stoichiometry of the complex of DOX
with Fol-c2-β-CyD in phosphate buffer (pH 7.3) at 25 °C was
determined by the continuous variation method,41 analyzing
the change in fluorescence intensity of DOX, where the total
concentrations of DOX and Fol-c2-β-CyD were kept constant
at 50 μM.
Stability Constant. The stability constants of the complex
of DOX with Fol-c2-β-CyD in phosphate buffer (pH 7.3 or 6.8)
were determined by the analysis of changes in fluorescence
intensity of DOX at λem 554 nm (λex 470 nm). The Kc values
were obtained from the following Scott’s equation,42 assuming
the 1:1 guest/host interaction
Synthesis of heptakis-6-NH2-cap2-β-CyD (NH2-c2-β-
CyD, 7). Boc-c2-β-CyD (21.86 g) was dissolved in 4 M HCl/
dioxane (85 mL) and the mixture was stirred for 4 h on ice.
After condensation, the reactant was lyophilized, and then
NH2-c2-β-CyD was obtained. The reaction was monitored by
TLC (silica gel F254, Merck, Whitehouse Station, NJ). Eluent:
1-buthanol/ethanol/water = 5:4:3 (v/v/v). Indicator: p-
anisaldehyde and ninhydrin. MALDI-TOF MS [M+Na]+ m/z
2735.
Synthesis of heptakis-6-Fol-cap2-β-CyD (Fol-c2-β-CyD,
8). NH2-c2-β-CyD (53.3 mg) and FA (276.9 mg) were
dissolved in DMSO (20 mL) at 90 °C. After cooling, NMM
(66 μL), DMT-MM (177.7 mg), and methanol (10 mL) were
added to the mixture, and then the mixture was stirred for 45 h
a·b·L/d = 1/(Kc·ε ) + b/ε
c
c
where a is the total concentration of DOX, b is the total
concentration of Fol-c2-β-CyD, L is length of cell, εc is the
difference in fluorescence intensity for free and complexed
DOX, and d is the change in fluorescence intensity of DOX by
the addition of Fol-c2-β-CyD.
Cell Culture. A549 cells, a human lung epithelium cell line
(FR-α (−)), were cultured as reported previously.43,44 KB cells,
a human squamous carcinoma cell line (FR-α (+)), and Colon-
26 cells, a mouse colon adenocarcinoma cell line (FR-α (+)),
were grown in a RPMI-1640 culture medium (FA-free)
containing penicillin (1 × 105 mU/mL) and streptomycin
C
dx.doi.org/10.1021/bc400015r | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Figure 1. Preparation pathways of Fol-c2-β-CyD.
(0.1 mg/mL) supplemented with 10% FBS at 37 °C in a
humidified 5% CO2 and 95% air atmosphere.
was measured with a microplate reader (Bio-Rad model 550,
Tokyo, Japan).
In Vitro Antitumor Activity. In vitro antitumor activity was
assayed by the WST-1 method (a Cell Counting Kit, Wako
Pure Chemical Industries, Osaka, Japan), as reported
previously.45,46 Briefly, KB cells and A549 cells were seeded
at 5 × 104 cells onto 96-well microplate (Iwaki, Tokyo, Japan)
and incubated for 24 h in a humidified atmosphere of 5% CO2
and 95% air at 37 °C. The cells were washed once with PBS
(pH 7.4). In the case of KB cells system, 150 μL of RPMI-1640
culture medium (FA-free) containing 10 μM DOX in the
absence and presence of Fol-c2-β-CyD (molar ratio = 1:1) or
Tween 20 was added to the cells and incubated for 24 h at 37
°C. In the case of A549 cells system, 150 μL of DMEM
containing 1 μM DOX in the absence and presence of Fol-c2-β-
CyD (molar ratio = 1:1) or Tween 20 was added to the cells
and incubated for 24 h at 37 °C. After washing once with PBS
to remove the samples, 100 μL of fresh Hanks’ balanced salt
solution (HBSS, pH 7.4) and 10 μL of WST-1 reagent were
added to the plates and incubated for 30 min at 37 °C. The
absorbance at 450 nm against a reference wavelength of 620 nm
Cellar Uptake of Fol-c2-β-CyD. KB cells and A549 cells
were seeded at 4 × 105 cells onto 35 mm microplate (Iwaki,
Tokyo, Japan), and incubated for 24 h in a humidified
atmosphere of 5% CO2 and 95% air at 37 °C. The cells were
washed once with 1 mL of PBS, and then incubated for 1 h at
37 °C with 2 mL of RPMI-1640 culture medium (FA-free) in
the KB cells system or DMEM in the A549 cells system in the
presence of 10 μM TRITC-labeled Fol-c2-β-CyD with or
without FA (4 mM). Next, the cells were washed with 1 mL of
PBS. Fluorescence intensity of TRITC was measured by
FACSCalibur (BD Biosciences, Franklin Lakes, NJ).
Cellar Uptake of DOX. KB cells were seeded at 4 × 105
cells onto 35 mm microplate (Iwaki, Tokyo, Japan), and
incubated for 24 h in a humidified atmosphere of 5% CO2 and
95% air at 37 °C. The cells were washed once with 1 mL of
PBS, and then incubated for 1 h at 37 °C with 1 mL of RPMI-
1640 culture medium (FA-free) containing 10 μM DOX or
DOX/Fol-c2-β-CyD (molar ratio = 1:1). After washing with 1
mL of RPMI-1640 culture medium, the cells were observed by
D
dx.doi.org/10.1021/bc400015r | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
a fluorescence microscopy BZ-9000 (KEYENCE, Osaka,
Japan).
by monitoring the fluorescence intensity change gave a
maximum peak at 0.5, indicating that Fol-c2-β-CyD forms an
inclusion complex with DOX at a 1:1 molar ratio.
Next, to determine the Kc of DOX/Fol-c2-β-CyD complex at
pH 7.3, fluorescence spectra of DOX in the absence and
presence of Fol-c2-β-CyD were measured (Figure 3A). Also, the
In Vivo Antitumor Effect. Four-week-old BALB/c male
mice (ca. 20 g) were subcutaneously injected with the
suspension containing Colon-26 carcinoma cells (2 × 105
cells/100 μL), FR-α-expressing cells. About 10 days later
(diameter of tumor was ca. 8 mm), the mannitol solution (5%)
including DOX (3 or 5 mg/kg) with or without Fol-c2-β-CyD
(25 mg/kg) was administered by single intratumoral or
intravenous injection to Colon-26 cells (FR-α (+))-bearing
mice. The tumor volume was determined by the equation
(Volume = LW2/2), where L is the longest dimension parallel
to the skin surface and W is the dimension perpendicular to L
and parallel to the surface. The body weight change of tumor-
bearing mice was monitored for 30 days.
Data Analysis. Data are given as the mean
SEM.
Statistical significance of mean coefficients for the studies was
performed by analysis of variance followed by Scheffe’s test. p-
Values for significance were set at 0.05.
RESULTS
■
Preparation of Fol-c2-β-CyD. Figure 1 shows the
preparation pathway of Fol-c2-β-CyD. First, per-NH2-β-CyD
was obtained through chlorination, and azidation of primary
hydroxyl groups of β-CyD. MALDI-TOF MS spectra of Cl-β-
CyD, N3-β-CyD, and NH2-β-CyD indicated that they have
seven chloro, azido, and amino groups, respectively (Figures
S1−3). Next, two caproic acids were condensed with amino
groups of NH2-β-CyD by DMT-MM (Figures S4, S5). After
deprotection (Figure S6), FA was modified to NH2-c2-β-CyD
using DMT-MM, and Fol-c2-β-CyD was obtained. Product
yield of Fol-c2-β-CyD was 34%, and no unreacted compounds
Figure 3. Effects of Fol-c2-β-CyD on fluorescence spectrum of DOX in
phosphate buffer (pH 7.3 or 6.8).
Kc value at pH 6.8 was determined (Figure 3B), since the pH
value in FR-α-mediated endosomes, i.e., GPI-anchored-protein-
enriched endosomal compartment (GEEC), is 6.0−6.8.47 The
fluorescence intensity of DOX decreased as the concentration
of Fol-c2-β-CyD increased, indicating a degree of interaction
between both compounds. Strikingly, the Kc values of the
complex determined using Scott’s equation were 2.4 × 106 M−1
at pH 7.3 (Table 1). It should be noted that the Kc value of the
1
were confirmed by TLC. According to the result of H NMR
spectrum, the degree of substitution of folate (DSF) was
determined to be 7.0 from the integral values of the protons of
benzene ring of folate and anomeric protons of glucose in β-
CyD (Figure S7A). In addition, MALDI-TOF MS spectrum of
Fol-c2-β-CyD showed a parent peak at 5698 m/z, indicating the
successful preparation of Fol-c2-β-CyD (DSF 7.0) (Figure
S7B).
Complex Formation between Fol-c2-β-CyD and DOX.
DOX is one of the most effective antitumor drugs. Hence, we
selected DOX as an antitumor drug in this study, and the
stoichiometry of a host−guest complex was determined by the
continuous variation plot method (Figure 2).41 The plots made
Table 1. Stability Constants (Kc) of DOX/β-CyDs
Complexes
β-CyD system
method
pH
Kc (M−1
)
Fol-c2β-CyD
Fol-c2-β-CyD
Fol-c2-β-CyD
FA-β-CyD
β-CyD
fluorescence spectrum
SPR
7.3
7.3
6.8
7.3
7.3
2.4 × 106
3.5 × 107
3.1 × 104
4.9 × 105
2.2 × 102
fluorescence spectrum
fluorescence spectrum
fluorescence spectrum
complex was also determined to be 3.5 × 107 M−1at pH 7.3 by
SPR method (Table 1). Interestingly, the Kc values of the
complexes of FA-β-CyD and β-CyD were 4.9 × 105 M−1 and
2.2 × 102 M−1, respectively, indicating that the extremely high
Kc value of the complex of Fol-c2-β-CyD with DOX at pH 7.3 is
reliable (Table 1). Meanwhile, the Kc value of DOX/Fol-c2-β-
CyD complex was 3.1 × 104 M−1 at pH 6.8 (Table 1). Hence,
these results suggest that Fol-c2-β-CyD forms the stable
complex with DOX in bloodstream, and the complex could
dissociate in endosomes after cellular uptake.
Figure 2. Continuous variation plots for DOX/Fol-c2-β-CyD system
monitored at 554 nm. The total concentration of DOX and Fol-c2-β-
Cellular Uptake. Cellular uptake of Fol-c2-β-CyD fluo-
rescence-labeled with TRITC was examined using both a
fluorescence microscope and flow cytometry. Strong fluo-
CyD was 50 μM. Each points represent the mean
SEM of 3
experiments.
E
dx.doi.org/10.1021/bc400015r | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
rescence of TRITC-Fol-c2-β-CyD was observed after treatment
for 1 h in KB cells, a FR-α-positive cell line (Figure S8). In
contrast, the fluorescence of TRITC-β-CyD was negligible,
suggesting that folate moieties in Fol-c2-β-CyD molecule
accelerate its uptake into KB cells. Furthermore, cellular uptake
of TRITC-Fol-c2-β-CyD in KB cells was significantly higher
than that in A549 cells, a FR-α-negative cell line (Figure 4A),
In Vitro Antitumor Activity. The antitumor activity of
DOX/Fol-c2-β-CyD complex was evaluated in KB cells (Figure
6A) and A549 cells (Figure 6B). The DOX concentration was
Figure 6. Cytotoxic activity of DOX/Fol-c2-β-CyD complex in KB
cells (FR-α (+)) (A) and A549 cells (FR-α (−)) (B) after treatment
for 24 h. The molar ratio of DOX/Fol-c2-β-CyD was 1:1. The
concentrations of DOX in the KB cells and A549 cells systems were 10
μM and 1 μM, respectively. Each value represents the mean SEM of
3−18 experiments. *p < 0.05, compared with DOX alone.
Figure 4. Cellular uptake of TRITC-Fol-c2-β-CyD into KB (FR-α (+))
cells and A549 cells (FR-α (−)) (A), and the effects of FA on cellular
uptake of TRITC-Fol-c2-β-CyD into KB cells (B). The fluorescence
intensity of TRITC in the cells was determined 1 h after incubation at
37 °C by a flow cytometer. The concentration of Fol-c2-β-CyD was 10
μM. The concentration of FA was 4 mM. Each value represents the
mean SEM of 3 experiments. *p < 0.05, compared with A549 cells
or TRITC-Fol-c2-β-CyD.
set at 10 μM and 1 μM in the KB cells and A549 cells,
respectively, because moderate antitumor activities of DOX
were observed at the concentration in both cells. Fol-c2-β-CyD
increased antitumor activity of DOX in KB cells, but not in
A549 cells. Importantly, these preferred effects of Fol-c2-β-CyD
were observed not only with DOX but also with other
hydrophobic antitumor drugs such as vinblastine (VBL) and
paclitaxel (PTX), whereas Fol-c2-β-CyD did not increase
antitumor activity of 5-FU, a hydrophilic antitumor drug
(Figure S10). These results suggest that Fol-c2-β-CyD could
increase antitumor activities of hydrophobic antitumor drugs
through markedly stable complexation, not hydrophilic drugs,
in FR-α-positive cells.
and was decreased by the addition of free FA as a competitor of
FR-α (Figure 4B). The amount of DOX in KB cells also
increased in the presence of Fol-c2-β-CyD (Figures 5, S9).
These results suggest that cellular uptake of Fol-c2-β-CyD or its
complex with DOX could be mediated by FR-α on KB cells.
In Vivo Antitumor Activity. To investigate the antitumor
activity of DOX/Fol-c2-β-CyD complex in vivo, the solution
containing the complex was administered intratumorally and
intravenously to mice subcutaneously inoculated with Colon-26
cells, an FR-α-positive mouse colon carcinoma cell line. As
shown in Figure 7A, an intratumoral administration of the
DOX/Fol-c2-β-CyD complex significantly inhibited the tumor
growth, compared to that of control and DOX alone.
Additionally, alterations in the body weight of the mice were
almost the same among the all samples (Figure 7B). Thus, Fol-
c2-β-CyD could increase antitumor activity of DOX after
intratumoral administration to tumor-bearing mice.
Figure 5. Cellular uptake of DOX into KB cells (FR-α (+)) in the
absence and presence of Fol-c2-β-CyD. The fluorescence intensity of
DOX in the cells was determined 1 h after incubation at 37 °C by a
fluorescence microscope. The concentration of Fol-c2-β-CyD and
Next, antitumor activity of DOX/Fol-c2-β-CyD complex after
single intravenous administration to tumor-bearing mice was
evaluated (Figure 7C). Intravenous injection of DOX did not
inhibit the tumor growth under the present experimental
DOX was 10 μM. Each value represents the mean
SEM of 3−6
experiments. *p < 0.05, compared with DOX alone.
F
dx.doi.org/10.1021/bc400015r | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
together, the Fol-c2-β-CyD/DOX complex stably exists in
bloodstream, and then may dissociate, at least in part, in GEEC
after cellular uptake through FR-α-mediated CLIC-GEEC
endocytic pathway.
Cellular uptake of Fol-c2-β-CyD in KB cells was higher than
that in A549 cells (Figure 4A). Likewise, a significantly high
amount of DOX was introduced into KB cells in the presence
of Fol-c2-β-CyD (Figure 5). Hattori et al. reported that
heptakis-galactose-branched β-CyD shows a high binding
constant with the asialoglycoprotein receptor, compared to
bis-galactose-branched β-CyD due to their strong multivalent
interaction.37 Thus, Fol-c2-β-CyD, possessing seven FA
molecules in a β-CyD molecule, could show the multivalent
interaction with FR-α. Meanwhile, a spacer between FA and a
carrier is important for recognition by FR-α, because a spacer
gives flexibility to the FA moiety.49 Most recently, we revealed
that cellular uptake of Fol-c2-β-CyD was twice as high as that of
Fol-c1-β-CyD, having one caproic acid as a spacer between FA
and a β-CyD molecule (data not shown). However, it is still
unknown whether Fol-c2-β-CyD has favorable length of spacer
and the DSF for cellular uptake into FR-α-positive cells.
Thereafter, we should prepare the various folate-appended β-
CyDs having a different length spacer, and evaluate their
binding ability to FR-α.
Fol-c2-β-CyD may suppress efflux of DOX from P-
glycoprotein (P-gp)-overexpressing cells through the complex-
ation. In the present study, cellular uptake of DOX was actually
increased twice by the addition of Fol-c2-β-CyD in KB cells
(Figure 5), and cellular efflux of DOX from KB cells was
significantly impaired by the complexation with Fol-c2-β-CyD
(Figure S11). Riganti et al. reported that DOX-containing
anionic liposomal nanoparticles was less extruded by P-gp than
DOX, and inhibited the pump activity.50 In addition, Roger et
al. demonstrated that FA functionalized nanoparticles have the
potential to enhance the oral absorption of drugs with poor oral
bioavailability involving in overcoming P-gp-mediated drug
efflux.51 These lines of evidence make it tempting to speculate
that Fol-c2-β-CyD may decrease the amount of P-gp in plasma
membrane and impairment of transport of P-gp. In fact, our
previous data that 2,6-di-O-methyl-β-CyD (DM-β-CyD)
decreased the P-gp level in Caco-2 cells and VBL-resistant
Caco-2 cells52 may support our speculation. Studies are
currently underway to investigate the mechanism by which
Fol-c2-β-CyD suppresses P-gp-mediated efflux of DOX in P-gp-
overexpressing cells.
Figure 7. Tumor volume (A,C) and body weight (B,D) after
intratumoral (A,B) and intravenous (C,D) administration of control
■
○
▲
( ), DOX ( ), and DOX/Fol-c2-β-CyD complex ( ) to Colon-26
cells (FR-α (+))-bearing mice. Each points represent the mean SEM
†
of 3−10 experiments. *p < 0.05, compared with control. p < 0.05,
compared with DOX alone.
conditions. In contrast, the DOX/Fol-c2-β-CyD complex
markedly inhibited the tumor growth after intravenous
injection with no significant alteration in the body weight of
the mice (Figure 7D). These results strongly suggest that Fol-
c2-β-CyD is useful for an antitumor drug carrier even after
intravenous administration.
DISCUSSION
■
In this study, we prepared folate-appended β-CyD with two
caproic acids as a spacer, and evaluated antitumor activities of
its complexes with DOX in vitro and in vivo. To our knowledge,
this is the first report in which β-CyD derivatives show a
systemic antitumor effect through not only its extremely stable
complexation with DOX, but also FA-dependent tumor
targeting after intravenous administration in tumor-bearing
mice.
Fol-c2-β-CyD increased antitumor activity of DOX in KB
cells, but did not do so in A549 cells (Figure 6). Similarly,
antitumor activities of VBL and PXT were increased by the
addition of Fol-c2-β-CyD in KB cells (Figures S10A, B).
Meanwhile, these effects were not observed with 5-FU (Figure
S10C). DOX, VBL, and PTX are hydrophobic drugs, which can
interact with CyDs. In contrast, 5-FU is a hydrophilic drug, and
thereby the interaction of 5-FU and β-CyD is very weak.53
Thus, a complexation ability of Fol-c2-β-CyD with antitumor
drugs such as DOX, VBL, PTX, and 5-FU plays an important
role for the antitumor effects of their complexes. Upadhyay et
al. reported that methyl-β-CyD enhances the susceptibility of
human breast cancer cells to carboplatin and 5-FU at high
concentration (5 mM).54 However, the concentration of Fol-c2-
β-CyD was only 10 μM in the present study. Therefore, it is
sure that Fol-c2-β-CyD does not enhance the susceptibility of
KB cells to 5-FU.
The complex of Fol-c2-β-CyD and DOX showed the
extremely high Kc value at pH 7.3 (∼106 M−1) (Table 1).
Hattori et al. reported that heptakis-sugar-branched CyDs with
caproic acids as a spacer between sugars and CyDs can strongly
interact with the hydrophobic drugs through “sea anemone
effect”, i.e., ligand−spacer moieties of the conjugates include
and intern the guest drugs.37 Therefore, Fol-c2-β-CyD could
also include and trap DOX through “sea anemone effect”. In
fact, β-CyD did not show the high Kc value with DOX (∼102
M−1) like the Fol-c2-β-CyD complex (Table 1). Importantly, it
is reported that a Kc value of more than 105 M−1 is required to
maintain a stable complex in vivo,33,48 signifying the potential of
Fol-c2-β-CyD as a drug carrier in vivo. In addition, the Kc value
of the complex of Fol-c2-β-CyD with DOX decreased to 3.1 ×
104 M−1 at pH 6.8, probably due to the ionization of amine
group in a DOX molecule. Yang et al. reported that the pH
value in the FR-α-mediated endosome is 6.0−6.8.47 Taken
G
dx.doi.org/10.1021/bc400015r | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Figure 8. Proposed scheme for improved antitumor effect of DOX by Fol-c2-β-CyD.
The complex of Fol-c2-β-CyD with DOX showed markedly
high antitumor activity after intratumoral and intravenous
administration to tumor-bearing mice (Figure 7). It is
noteworthy that this complex was administrated as a single
dose, although the antitumor activity continued over 30 days.
As shown in Figure S7B, the molecular weight of Fol-c2-β-CyD
is 5698, and is not sufficient for avoidance of glomerular
filtration. In addition, Fol-c2-β-CyD and DOX did not form any
aggregates in phosphate buffer (data not shown). Soliman et al.
demonstrated the presence of two folate-binding sites per
albumin molecule and the equilibrium constant of ∼103 M−1.55
Moreover, FA strongly binds to folate binding protein (FBP) in
blood circulation.56 Hence, the complex of Fol-c2-β-CyD and
DOX may bind to albumin and/or FBP, resulting in its long
circulating half-life. Thereafter, pharmacokinetics of the
complex of Fol-c2-β-CyD and DOX after intravenous
administration in mice should be clarified.
On the basis of the results obtained in this study, a proposed
mechanism for improved antitumor effect of DOX by Fol-c2-β-
CyD is shown in Figure 8. Fol-c2-β-CyD forms stable
complexes with DOX and the other hydrophobic antitumor
drugs in aqueous solution. After intravenous administration to
tumor-bearing mice, the complex may accumulate in the tumor
and may be incorporated in tumor cells through FR-α-mediated
endocytosis. In addition, the complex may dissociate in
endosome due to a decrease in the interaction between Fol-
c2-β-CyD and DOX. Finally, DOX may intercalate into DNA
and inhibit the reaction of topoisomerase II, showing the
antitumor activity (Figure 8). As described above, further
investigations regarding the safety profile of the DOX complex
with Fol-c2-β-CyD as well as the detailed mechanism by which
Fol-c2-β-CyD enhanced antitumor activity of DOX are
required.
CONCLUSION
■
In the present study, Fol-c2-β-CyD was successfully prepared,
and increased antitumor activities of antitumor drugs such as
DOX, VBL, and PTX, not 5-FU in vitro and in vivo. In addition,
Fol-c2-β-CyD can very easily form complexes with antitumor
drugs by simple mixing in aqueous solution. These results
suggest that Fol-c2-β-CyD could potentially be used as a
promising antitumor drug carrier in vitro and in vivo, especially a
systemic administration.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and supporting figures. This material
■
S
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +81-96-371-4160. Fax: +81-96-371-4420. E-mail:
arimah@gpo.kumamoto-u.ac.jp.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was financially supported by the Sasagawa
Scientific Research Grant from The Japan Science Society (R.
Onodera, 2012).
ABBREVIATIONS
■
FA, folic acid; CyD, cyclodextrin; DDS, drug delivery system;
EPR, enhanced permeability and retention; PEG, polyethylene
glycol; DOX, doxorubicin; VBL, vinblastine; PTX, paclitaxel; 5-
FU, 5-fluorouracil; RES, reticuloendothelial system; FR-α,
folate receptor-α; NMM, N-methylmorpholine; DMT-MM, 4-
(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride; DSF, degree of substitution of folate; SPR, surface
H
dx.doi.org/10.1021/bc400015r | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
folate-targeted liposomal doxorubicin in folate receptor-expressing
tumor models. Cancer Chemother. Pharmacol. 66, 43−52.
(18) Riviere, K., Huang, Z., Jerger, K., Macaraeg, N., and Szoka, F. C.,
Jr. (2011) Antitumor effect of folate-targeted liposomal doxorubicin in
KB tumor-bearing mice after intravenous administration. J. Drug
Targeting 19, 14−24.
(19) Watanabe, K., Kaneko, M., and Maitani, Y. (2012) Functional
coating of liposomes using a folate-polymer conjugate to target folate
receptors. Int. J. Nanomed. 7, 3679−3688.
(20) Wang, Y., Cao, X., Guo, R., Shen, M., Zhang, M., Zhu, M., and
Shi, X. (2011) Targeted delivery of doxorubicin into cancer cells using
a folic acid-dendrimer conjugate. Polym. Chem. 2, 1754−1760.
(21) Sahu, S. K., Mallick, S. K., Santra, S., Maiti, T. K., Ghosh, S. K.,
and Pramanik, P. (2010) In vitro evaluation of folic acid modified
carboxymethyl chitosan nanoparticles loaded with doxorubicin for
targeted delivery. J. Mater. Sci. Mater. Med. 21, 1587−1597.
(22) Scomparin, A., Salmaso, S., Bersani, S., Satchi-Fainaro, R., and
Caliceti, P. (2011) Novel folated and non-folated pullulan
bioconjugates for anticancer drug delivery. Eur. J. Pharm. Sci. 42,
547−558.
(23) Kim, D., Lee, E. S., Oh, K. T., Gao, Z. G., and Bae, Y. H. (2008)
Doxorubicin-loaded polymeric micelle overcomes multidrug resistance
of cancer by double-targeting folate receptor and early endosomal pH.
Small 4, 2043−2050.
(24) Leamon, C. P., Reddy, J. A., Vlahov, I. R., Westrick, E., Parker,
N., Nicoson, J. S., and Vetzel, M. (2007) Comparative preclinical
activity of the folate-targeted Vinca alkaloid conjugates EC140 and
EC145. Int. J. Cancer 121, 1585−1592.
plasmon resonance; TRITC, tetramethyl rhodamine isothio-
cyanate; P-gp, P-glycoprotein; FBP, folate binding protein
REFERENCES
■
(1) Danhier, F., Feron, O., and Preat, V. (2010) To exploit the tumor
microenvironment: Passive and active tumor targeting of nanocarriers
for anti-cancer drug delivery. J. Controlled Release 148, 135−146.
(2) Matsumura, Y., and Maeda, H. (1986) A new concept for
macromolecular therapeutics in cancer chemotherapy: mechanism of
tumoritropic accumulation of proteins and the antitumor agent
smancs. Cancer Res. 46, 6387−6392.
(3) Talekar, M., Kendall, J., Denny, W., and Garg, S. (2011)
Targeting of nanoparticles in cancer: drug delivery and diagnostics.
Anticancer Drugs 22, 949−962.
(4) Hong, R. L., Huang, C. J., Tseng, Y. L., Pang, V. F., Chen, S. T.,
Liu, J. J., and Chang, F. H. (1999) Direct comparison of liposomal
doxorubicin with or without polyethylene glycol coating in C-26
tumor-bearing mice: is surface coating with polyethylene glycol
beneficial? Clin. Cancer Res. 5, 3645−3652.
(5) Maruyama, K. (2011) Intracellular targeting delivery of liposomal
drugs to solid tumors based on EPR effects. Adv. Drug Delivery Rev. 63,
161−169.
(6) Schiffelers, R. M., Koning, G. A., ten Hagen, T. L., Fens, M. H.,
Schraa, A. J., Janssen, A. P., Kok, R. J., Molema, G., and Storm, G.
(2003) Anti-tumor efficacy of tumor vasculature-targeted liposomal
doxorubicin. J. Controlled Release 91, 115−122.
(7) Roche, A. C., Fajac, I., Grosse, S., Frison, N., Rondanino, C.,
Mayer, R., and Monsigny, M. (2003) Glycofection: facilitated gene
transfer by cationic glycopolymers. Cell. Mol. Life Sci. 60, 288−297.
(8) Hatakeyama, H., Akita, H., Maruyama, K., Suhara, T., and
Harashima, H. (2004) Factors governing the in vivo tissue uptake of
transferrin-coupled polyethylene glycol liposomes in vivo. Int. J. Pharm.
281, 25−33.
(9) Miyajima, Y., Nakamura, H., Kuwata, Y., Lee, J. D., Masunaga, S.,
Ono, K., and Maruyama, K. (2006) Transferrin-loaded nido-carborane
liposomes: tumbetaor-targeting boron delivery system for neutron
capture therapy. Bioconjugate Chem. 17, 1314−1320.
(10) Harata, M., Soda, Y., Tani, K., Ooi, J., Takizawa, T., Chen, M.,
Bai, Y., Izawa, K., Kobayashi, S., Tomonari, A., Nagamura, F.,
Takahashi, S., Uchimaru, K., Iseki, T., Tsuji, T., Takahashi, T. A.,
Sugita, K., Nakazawa, S., Tojo, A., Maruyama, K., and Asano, S. (2004)
CD19-targeting liposomes containing imatinib efficiently kill Phila-
delphia chromosome-positive acute lymphoblastic leukemia cells.
Blood 104, 1442−1449.
(11) Sugahara, K. N., Teesalu, T., Karmali, P. P., Kotamraju, V. R.,
Agemy, L., Girard, O. M., Hanahan, D., Mattrey, R. F., and Ruoslahti,
E. (2009) Tissue-penetrating delivery of compounds and nanoparticles
into tumors. Cancer Cell 16, 510−520.
(12) Sugahara, K. N., Teesalu, T., Karmali, P. P., Kotamraju, V. R.,
Agemy, L., Greenwald, D. R., and Ruoslahti, E. (2010) Coadministra-
tion of a tumor-penetrating peptide enhances the efficacy of cancer
drugs. Science 328, 1031−1035.
(13) Chen, H., Ahn, R., Van den Bossche, J., Thompson, D. H., and
O’Halloran, T. V. (2009) Folate-mediated intracellular drug delivery
increases the anticancer efficacy of nanoparticulate formulation of
arsenic trioxide. Mol. Cancer Ther. 8, 1955−1963.
(14) Lu, Y., and Low, P. S. (2002) Folate-mediated delivery of
macromolecular anticancer therapeutic agents. Adv. Drug Delivery Rev.
54, 675−693.
(15) Leamon, C. P., and Low, P. S. (2001) Folate-mediated targeting:
from diagnostics to drug and gene delivery. Drug Discovery Today 6,
44−51.
(16) Parker, N., Turk, M. J., Westrick, E., Lewis, J. D., Low, P. S., and
Leamon, C. P. (2005) Folate receptor expression in carcinomas and
normal tissues determined by a quantitative radioligand binding assay.
Anal. Biochem. 338, 284−293.
(17) Gabizon, A., Tzemach, D., Gorin, J., Mak, L., Amitay, Y.,
Shmeeda, H., and Zalipsky, S. (2010) Improved therapeutic activity of
(25) Hirayama, F., and Uekama, K. (1999) Cyclodextrin-based
controlled drug release system. Adv. Drug Delivery Rev. 36, 125−141.
(26) Li, J. J., Zhao, F., and Li, J. (2011) Supramolecular polymers
based on cyclodextrins for drug and gene delivery. Adv. Biochem. Eng.
Biotechnol. 125, 207−249.
(27) Szejtli, J. (1994) Medicinal applications of cyclodextrins. Med.
Res. Rev. 14, 353−386.
(28) Szente, L., and Szejtli, J. (1999) Highly soluble cyclodextrin
derivatives: chemistry, properties, and trends in development. Adv.
Drug Delivery Rev. 36, 17−28.
(29) Uekama, K., Hirayama, F., and Irie, T. (1998) Cyclodextrin drug
carrier systems. Chem. Rev. 98, 2045−2076.
(30) Salmaso, S., Semenzato, A., Caliceti, P., Hoebeke, J., Sonvico, F.,
Dubernet, C., and Couvreur, P. (2004) Specific antitumor targetable β-
cyclodextrin-poly(ethylene glycol)-folic acid drug delivery bioconju-
gate. Bioconjugate Chem. 15, 997−1004.
(31) Caliceti, P., Salmaso, S., Semenzato, A., Carofiglio, T., Fornasier,
R., Fermeglia, M., Ferrone, M., and Pricl, S. (2003) Synthesis and
physicochemical characterization of folate-cyclodextrin bioconjugate
for active drug delivery. Bioconjugate Chem. 14, 899−908.
(32) Zhang, H., Cai, Z., Sun, Y., Yu, F., Chen, Y., and Sun, B. (2012)
Folate-conjugated β-cyclodextrin from click chemistry strategy and for
tumor-targeted drug delivery. J. Biomed. Mater. Res. A 100, 2441−2449.
(33) Stella, V. J., and He, Q. (2008) Cyclodextrins. Toxicol. Pathol.
36, 30−42.
(34) Ooya, T., Eguchi, M., and Yui, N. (2003) Supramolecular design
for multivalent interaction: maltose mobility along polyrotaxane
enhanced binding with concanavalin A. J. Am. Chem. Soc. 125,
13016−13017.
(35) Ooya, T., Utsunomiya, H., Eguchi, M., and Yui, N. (2005) Rapid
binding of concanavalin A and maltose-polyrotaxane conjugates due to
mobile motion of α-cyclodextrins threaded onto a poly(ethylene
glycol). Bioconjugate Chem. 16, 62−69.
(36) Abe, H., Kenmoku, A., Yamaguch, N., and Hattori, K. (2002)
Structural effects of oligosaccharide-branched cyclodextrins on the
dual recognition toward lectin and drug. J. Incl. Phenom. Macrocycl.
Chem. 44, 39−47.
(37) Hattori, K., Kenmoku, A., Mizuguchi, T., Ikeda, D., Mizuno, M.,
and Inazu, T. (2006) Saccharide-branched Cyclodextrins as Targeting
Drug Carriers. J. Incl. Phenom. Macrocycl. Chem. 56, 9−16.
I
dx.doi.org/10.1021/bc400015r | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
(38) Oda, Y., Kobayashi, N., Yamanoi, T., Katsuraya, K., Takahashi,
K., and Hattori, K. (2008) β-Cyclodextrin conjugates with glucose
moieties designed as drug carriers: their syntheses, evaluations using
concanavalin A and doxorubicin, and structural analyses by NMR
spectroscopy. Med. Chem. 4, 244−255.
(56) Antony, A. C. (1992) The biological chemistry of folate
receptors. Blood 79, 2807−2820.
(39) Oda, Y., Yanagisawa, H., Maruyama, M., Hattori, K., and
Yamanoi, T. (2008) Design, synthesis and evaluation of D-galactose-β-
cyclodextrin conjugates as drug-carrying molecules. Bioorg. Med. Chem.
16, 8830−8840.
(40) Yamanoi, T., Yoshida, N., Oda, Y., Akaike, E., Tsutsumida, M.,
Kobayashi, N., Osumi, K., Yamamoto, K., Fujita, K., Takahashi, K., and
Hattori, K. (2005) Synthesis of mono-glucose-branched cyclodextrins
with a high inclusion ability for doxorubicin and their efficient
glycosylation using Mucor hiemalis endo-β-N-acetylglucosaminidase.
Bioorg. Med. Chem. Lett. 15, 1009−1013.
(41) Job, P. (1928) Formation and stability of inorganic complexes in
solution. Ann. Chim. 9, 113−203.
(42) Scott, R. L. (1956) Some comments on the Benesi-Hildebrand
equation. Rec. Trav. Chim. 75, 787−789.
(43) Arima, H., Chihara, Y., Arizono, M., Yamashita, S., Wada, K.,
Hirayama, F., and Uekama, K. (2006) Enhancement of gene transfer
activity mediated by mannosylated dendrimer/α-cyclodextrin con-
jugate (generation 3, G3). J. Controlled Release 116, 64−74.
(44) Wada, K., Arima, H., Tsutsumi, T., Chihara, Y., Hattori, K.,
Hirayama, F., and Uekama, K. (2005) Improvement of gene delivery
mediated by mannosylated dendrimer/α-cyclodextrin conjugates. J.
Controlled Release 104, 397−413.
(45) Hamasaki, K., Kogure, K., and Ohwada, K. (1996) A biological
method for the quantitative measurement of tetrodotoxin (TTX):
tissue culture bioassay in combination with a water-soluble tetrazolium
salt. Toxicon 34, 490−495.
(46) Ono, N., Arima, H., Hirayama, F., and Uekama, K. (2001) A
moderate interaction of maltosyl-α-cyclodextrin with Caco-2 cells in
comparison with the parent cyclodextrin. Biol. Pharm. Bull. 24, 395−
402.
(47) Yang, J., Chen, H., Vlahov, I. R., Cheng, J. X., and Low, P. S.
(2007) Characterization of the pH of folate receptor-containing
endosomes and the rate of hydrolysis of internalized acid-labile folate-
drug conjugates. J. Pharmacol. Exp. Ther. 321, 462−468.
(48) Stella, V. J., Rao, V. M., Zannou, E. A., and Zia, V. V. (1999)
Mechanisms of drug release from cyclodextrin complexes. Adv. Drug
Delivery Rev. 36, 3−16.
(49) Ghaghada, K. B., Saul, J., Natarajan, J. V., Bellamkonda, R. V.,
and Annapragada, A. V. (2005) Folate targeting of drug carriers: a
mathematical model. J. Controlled Release 104, 113−128.
(50) Riganti, C., Voena, C., Kopecka, J., Corsetto, P. A., Montorfano,
G., Enrico, E., Costamagna, C., Rizzo, A. M., Ghigo, D., and Bosia, A.
(2011) Liposome-encapsulated doxorubicin reverses drug resistance
by inhibiting P-glycoprotein in human cancer cells. Mol. Pharm. 8,
683−700.
(51) Roger, E., Kalscheuer, S., Kirtane, A., Guru, B. R., Grill, A. E.,
Whittum-Hudson, J., and Panyam, J. (2012) Folic acid functionalized
nanoparticles for enhanced oral drug delivery. Mol. Pharm. 9, 2103−
2110.
(52) Yunomae, K., Arima, H., Hirayama, F., and Uekama, K. (2003)
Involvement of cholesterol in the inhibitory effect of dimethyl-β-
cyclodextrin on P-glycoprotein and MRP2 function in Caco-2 cells.
FEBS Lett. 536, 225−231.
(53) Wen, J. Q., and Cui, W. (1990) Studies on the composition and
stability constant of inclusion complexes of β-cyclodextrin with
fluorouracil and ftorafur by NMR. Yao Xue Xue Bao 25, 345−348.
(54) Upadhyay, A. K., Singh, S., Chhipa, R. R., Vijayakumar, M. V.,
Ajay, A. K., and Bhat, M. K. (2006) Methyl-β-cyclodextrin enhances
the susceptibility of human breast cancer cells to carboplatin and 5-
fluorouracil: involvement of Akt, NF-κB and Bcl-2. Toxicol. Appl.
Pharmacol. 216, 177−185.
(55) Soliman, H. A., and Olesen, H. (1976) Folic acid binding by
human plasma albumin. Scand. J. Clin. Lab. Invest. 36, 299−304.
J
dx.doi.org/10.1021/bc400015r | Bioconjugate Chem. XXXX, XXX, XXX−XXX