A medusa-like β-cyclodextrin with 1-methyl-2-(2'-carboxyethyl) maleic anhydrides, a potential carrier for pH-sensitive drug delivery

Article abstract of DOI:10.3109/1061186X.2014.928718

We developed a new pH-sensitive drug delivery carrier based on β-cyclodextrin (β-CD) and 1-methyl-2-(2'-carboxyethyl) maleic anhydrides (MCM). The primary hydroxyl groups of β-CD were successfully attached to MCM residues to produce a medusa-like β-CD-MCM. The MCM residue was conjugated with cephradine (CP) with high efficiency (>90%). More importantly, β-CD-MCM-CP responded to the small pH drop from 7.4 to 5.5 and released greater than 80% of the drugs within 0.5h at pH 5.5. In addition, the inclusion complex between β-CD-MCM-CP and the adamantane derivative was formed by simple mixing to show the possibility of introducing multi-functionality. Based on these results, β-CD-MCM can target weakly acidic tissues or organelles, such as tumours, inflammatory tissues, abscesses or endosomes, and be easily modified with various functional moieties, such as ligands for cell binding or penetration, enabling more efficient and specific drug delivery.

Full text of DOI:10.3109/1061186X.2014.928718

http://informahealthcare.com/drt
ISSN: 1061-186X (print), 1029-2330 (electronic)
J Drug Target, 2014; 22(7): 658–668
2014 Informa UK Ltd. DOI: 10.3109/1061186X.2014.928718
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ORIGINAL ARTICLE
0
A medusa-like b-cyclodextrin with 1-methyl-2-(2 -carboxyethyl) maleic
anhydrides, a potential carrier for pH-sensitive drug delivery
1
1
1
1
2
1
Sunyoung Kang , Euddeum Park , Youngeun Kim , Seonju Lee , Jiwoong Kwon , Hyungdo Cho , and Yan Lee
1
1 2
Department of Chemistry and WCU Department of Biophysics and Chemical Biology, College of Natural Sciences, Seoul National University,
Seoul, Republic of Korea
Abstract
Keywords
We developed a new pH-sensitive drug delivery carrier based on b-cyclodextrin (b-CD) and
Acidic environment, cephradine,
dimethylmaleic acid anhydride, drug
conjugation
0
1
-methyl-2-(2 -carboxyethyl) maleic anhydrides (MCM). The primary hydroxyl groups of b-CD
were successfully attached to MCM residues to produce a medusa-like b-CD–MCM. The MCM
residue was conjugated with cephradine (CP) with high efficiency (490%). More importantly,
b-CD–MCM–CP responded to the small pH drop from 7.4 to 5.5 and released greater than 80%
of the drugs within 0.5 h at pH 5.5. In addition, the inclusion complex between b-CD–MCM–CP
and the adamantane derivative was formed by simple mixing to show the possibility of
introducing multi-functionality. Based on these results, b-CD–MCM can target weakly acidic
tissues or organelles, such as tumours, inflammatory tissues, abscesses or endosomes, and be
easily modified with various functional moieties, such as ligands for cell binding or penetration,
enabling more efficient and specific drug delivery.
History
Received 1 April 2014
Revised 14 May 2014
Accepted 22 May 2014
Published online 23 June 2014
Introduction
should be delivered into the cell interior via an endocytic
pathway [18]. However, smart materials with rapid degrad-
ability responding to small pH variations are still under active
investigation for a more practical drug delivery system
because the pH variation in the human body is not extreme,
with a few exceptions.
For efficient and safe delivery of drugs to specific targets,
smart drug delivery techniques have evolved along with the
development of various signal-responsive materials [1–3].
Abrupt changes in the physicochemical properties of smart
materials in response to physical signals such as temperature
As a chemical moiety for pH-responsive smart materials,
maleic acid amide derivatives are attractive owing to their
rapid degradability at an appropriate pH. Unlike other amides,
which can only be degraded at extreme pH conditions, maleic
acid amide derivatives can be degraded within a biologically
tolerable pH range [19]. The responsive pH range and the
degradation rate can be finely tuned by a change of alkyl
substituents on the cis-double bonds [20]. Moreover, the
maleic acid amide derivatives can be synthesised via the one-
step ring-opening reaction between amines and corresponding
maleic acid anhydride derivatives under weakly alkaline
conditions. The easy synthesis of maleic acid amide deriva-
tives under mild conditions is a strong advantage over other
pH-sensitive linkages, which require quite complicated syn-
thetic steps in conjugation of drugs or cross-linking of carrier
backbones [21].
[
4] and light [5] as well as chemical signals such as pH [6],
glutathione [7], and enzymes [8] can induce encapsulation
and release of drugs in specific conditions. Among these, pH
has been frequently selected as the controlling trigger because
the pH value can represent a certain physiological condition
in many target sites in the body. The pH variation through the
gastrointestinal tract has been used for the site-specific drug
delivery to strongly acidic stomach or weakly alkaline small
intestine [9]. Enhanced glycolysis in cancer tissues lowers the
pH to 5–6, and similar pH lowering is also observed in
inflammatory tissues [10] and abscesses [11,12]. The weakly
acidic pH value is recognised as an inducing signal for the
release of anti-cancer [13] or anti-inflammatory drugs [14].
Moreover, the early endosomal pH value of 5–6 is frequently
used as the signal for the endosomal escape of macromol-
ecular drugs such as pDNA [15], siRNA [16] or proteins [17],
which have difficulty penetrating the cell membrane and
In this study, we wanted to synthesise a drug carrier based
0
on 1-methyl-2-(2 -carboxyethyl) maleic anhydride (MCM) or
carboxylate dimethyl maleic anhydride (CDM), a maleic acid
anhydride derivative with two alkyl substituents. Because the
acid amide correspondent from MCM is stable at normal
physiological pH 7.4 but rapidly degradable at pH 5.5 [22],
an MCM derivative might be one of the best candidates for
the conjugation with drugs targeting weakly acidic
Address for correspondence: Yan Lee, Department of Chemistry,
College of Natural Sciences, Seoul National University, Gwanak-ro 1,
Gwanak-gu, Seoul 151-747, Republic of Korea. Tel: +82 2 880 4344.
Fax: +82 2 871 2496. E-mail: gacn@snu.ac.kr

DOI: 10.3109/1061186X.2014.928718
A medusa-like b-CD–MCM 659
environments. We designed a drug carrier with several MCM
residues on the surface for the easiest conjugation with amine-
based drugs or cross-linkers.
Materials and methods
Materials
As the backbone of the drug carrier, we selected
b-cyclodextrin (b-CD), a cyclic oligosaccharide with seven
glucose molecules. b-Cyclodextrin (b-CD) has been widely
used as a part of drug delivery carrier due to its high
biocompatibility [23]. Many functional groups can be readily
introduced into the b-CD backbone because it has 7 primary
and 14 secondary hydroxyl groups. A medusa-like structure
can be synthesised by reaction with the seven primary
hydroxyl groups [24]. The regioselective functionalization
of b-CD enables to achieve homogeneous chemical properties
of the conjugated functional moieties. Furthermore, the
toroidal interior of b-CD can be used for the introduction of
additional functionality such as a targeting ligand [25] or cell-
penetrating moiety [26] by host–guest inclusion because the
interior can strongly interact with small hydrophobic mol-
ecules such as adamantane via non-covalent interactions [27].
Polymers or nanoparticles with adamantane moieties can be
easily modified with b-CD derivatives by the host–guest
interaction [28].
CP was obtained from Han Wha Pharm. Co., Ltd. (Seoul,
South Korea). b-CD, 6-aminohexanoic acid, di-tert-butyl
dicarbonate ((Boc) O), n-butylamine (BA), triethylamine
2
(TEA), diisopropylethylamine (DIPEA), diisopropylcarbodii-
mide (DIC), 4-dimethylaminopyridine (DMAP), p-toluene-
sulphonic acid (PTSA), trifluoroacetic acid (TFA), sodium
0
0
hydride (NaH) (60% in mineral oil), N,N,N ,N -tetramethyl-O-
(1H-benzotriazol-1-yl)uronium hexafluorophosphate
(HBTU), fluorescein isothiocyanate (FITC) isomer I and
dimethylsulphoxide (DMSO) were purchased from Sigma-
Aldrich (St Louis, MO). Triethyl-2-phosphonopropionate,
dimethyl-2-oxoglutarate and 1-adamantaneamine hydrochlor-
ide were purchased from TCI (Tokyo, Japan). Ammonium
chloride (NH
hydroxide (KOH), sodium chloride (NaCl), sodium hydroxide
(NaOH), sodium bicarbonate (NaHCO ), tetrahydrofuran
Cl), magnesium sulphate (MgSO ), potassium
4
4
3
(THF), dimethylformamide (DMF), hexane, ethyl acetate
(EA), methanol (MeOH), ethanol, dichloromethane (DCM),
hydrochloric acid (HCl), acetonitrile (ACN) and pyridine
were purchased from Daejung (Seoul, South Korea).
Anhydrous TEA, THF and DMF were obtained by distillation
of the reagent-grade materials. Other reagents were used
without further purification.
We expected that the medusa-like b-CD could be
conjugated with drug molecules via pH-sensitive MCM
linkers by simple mixing in mildly alkaline conditions and
that other functional moieties with adamantane residues could
also be non-covalently introduced into the pore of the b-CD.
Otherwise, the surface of other drug carriers such as polymers
or nanoparticles can be non-covalently modified with the
pH-sensitive medusa-like b-CD derivative. The resulting
carrier could rapidly release the conjugated drugs in weakly
acidic conditions. The general conceptual scheme is illu-
strated in Figure 1. By using cephradine (CP), a cephalosporin
antibiotic, as a drug to be targeted to weakly acidic
inflammatory tissues, we wanted to prove our concept
in vitro. The medusa-like b-CD derivatives has strong
potential for multi-functional drug delivery systems for
targeting tumours, inflammatory tissues or abscesses as well
as for the rapid response at the pH of early endosomes in
intracellular drug delivery.
Synthesis
Synthesis of compound 1. Following the method described in
a previous report [29], compound 1 was prepared. Briefly,
NaH (0.37 g, 9.2 mmol) was added slowly into a solution of
triethyl-2-phosphonopropionate (1.64 g, 6.89 mmol) in anhyd-
ꢀ
rous THF (30 mL) at 0 C under a nitrogen atmosphere.
Dimethyl-2-oxoglutarate (1.00 g, 5.74 mmol) was added to the
solution after the evolution of hydrogen gas had stopped. The
reaction mixture was further stirred while maintaining the
ꢀ
temperature at 0 C. After the reaction completion was
confirmed by TLC, a saturated aqueous solution of NH Cl
was added dropwise. Following the removal of THF by rotary
4
Figure 1. General scheme of pH-sensitive drug delivery by a medusa-like b-CD–MCM.

6
60 S. Kang et al.
J Drug Target, 2014; 22(7): 658–668
evaporation, the resulting solid and water mixture was
extracted with EA several times. The organic phase was
combined, washed with deionised water (DIW) and brine,
for compound 4, but using CP solution (6 equiv.) in ACN
instead of BA. After purification, yellow solid was obtained.
1
Yield:490%, H NMR (300 MHz, 1.1 wt% NaOD in D O): d
2
dried over MgSO and concentrated by rotary evaporation.
4
1.83 (3H, s, CH -cephalosporin), 1.86 (3H, s, CCH ), 2.18–
2.24 (2H, m, CCH H ), 2.45–2.48 (2H, m, CH CH CO),
2 2 2 2
3
3
The residue was purified by silica gel chromatography eluted
with EA/hexane to yield compound 1 as a colourless oil.
0
0
2.62–2.71 (4H, m, 3 , 6 -CH -cyclohexadiene), 2.99–3.08
2
1
Yield: 91%, H NMR (300 MHz, CDCl ): d 1.25–1.30
(1H, m, SCH C-cephalosporin), 3.12–3.18 (1H, m, SCH C-
2
3
2
0 0
(
CCH CH ), 2.62–2.65 (2H, m, CH CH CO), 3.66 (3H, s,
3H, t, CH CH O), 1.98 (3H, s, CH C), 2.46–2.49 (2H, m,
cephalosporin), 3.39–3.49 (2H, m, 2 , 4 -CH-b-CD), 3.77–
3
2
3
0
0
0
0
3.83 (4H, m, 3 , 5 -CH, 6 -CH -b-CD), 4.73 (1H, s, 1 -CCH-
2
2
2
2
2
0
CO CH ), 3.73 (3H, s, CH O CC) and 4.16–4.22 (2H, q,
3 2
cyclohexadiene), 4.87 (1H, s, NCHS), 4.90 (1H, s, 1 -CH-
0
2
3
0
CH CH O).
3
b-CD), 5.71–5.73 (3H, m, NHCHCO, 4 , 5 -CH-cyclohex-
2
0
Synthesis of compound 2. Following the method described
in a previous report [29], compound 2 was prepared. Briefly,
compound 1 in a 2 M KOH solution in ethanol was allowed to
reflux for 1 h. DIW was added, and the hot reaction mixture
was cooled to ambient temperature. After removal of ethanol
by evaporation, the aqueous phase was washed with DCM
several times and acidified to pH 2 using concentrated HCl.
The aqueous phase was then extracted with EA several times.
adiene), 5.87 (1H, s, 2 -CH-cyclohexadiene).
Synthesis of compound 6. 6-Aminohexanoic acid (1.00 g,
7.26 mmol) was dissolved in the co-solvent of THF:aqueous
saturated NaHCO3 solution (1:1) and a solution of di-
tert-butyl dicarbonate (2.00 g, 9.15 mmol) in THF was
added dropwise into the solution at ambient temperature.
After the reaction completion was confirmed by TLC,
following the removal of THF by rotary evaporation and the
resulting solution was acidified down to pH 1–2. The mixture
was extracted with DCM several times. The organic phase
was combined, washed with deionised brine, dried over
The organic phase was dried over MgSO and concentrated
4
under reduced pressure to produce MCM (2) as a white solid.
1
Yield: 74%, H NMR (300 MHz, CDCl ): d 2.12 (3H, s,
3
CCH ) and 2.77 (4H, s, CCH CH CO).
3
MgSO and concentrated by rotary evaporation. The pure
4
2
2
Synthesis of compound 3. To a solution of compound 2
2.00 g, 1.09 mmol), b-CD (0.10 g, 0.092 mmol), DMAP
0.46 g, 0.37 mmol) and PTSA (0.70 g, 0.37 mmol) in DMF
10 mL) was added DIC (0.33 g, 2.6 mmol). The reaction
compound, N-Boc-aminohexanoic acid (6) was obtained as an
1
opaque crystal. Yield: 81%, H NMR (300 MHz, 1.1 wt%
(
(
(
NaOD in D O): d 1.10–1.28 (2H, m, CH CH CH ), 1.32–1.56
2
2
2
2
(13H, m, CH CH CH CH CH , t-Boc), 2.10–2.15 (2H, t,
2
2
2
2
2
mixture was stirred overnight at ambient temperature. The
crude product was purified by dialysis with regenerated
CH CH CO H) and 2.99–3.03 (2H, t, NHCH CH ).
2 2 2 2 2
Synthesis of compound 7. Following the method described
in a previous report [30], compound 7 was prepared. Briefly, a
solution of compound 6 (0.10 g, 0.43 mmol) and 1-adamanta-
neamine hydrochloride (0.080 g, 0.43 mmol) in dry THF
(9 mL) was treated with DIPEA (0.12 mL, 0.86 mmol) and
HBTU (0.16 g, 0.43 mmol). After being stirred for 16 h at
ꢀ
cellulose membrane (MWCO 1000, SPECTRUM ) in phos-
phate buffer (pH 9.0), HCl solution (pH 3.0) and DIW,
respectively. Lyophilisation was performed next. An off-white
1
power, b-CD–MCM (3) was obtained. Yield: 85%, H NMR
(
300 MHz, 1.1 wt% NaOD in D O): d 1.65 (3H, s, CCH ),
2
3
ꢀ
1
.98–2.10
(2H, m,
CCH CH ),
2
2.21–2.33
(2H, m,
room temperature, the reaction mixture was heated at 60 C
2
0
0
CH CH CO), 3.26–3.36 (2H, m, 2 , 4 -CH-b-CD), 3.67–3.80
for 90 min. Then, DCM and brine were added, and the organic
phase was washed twice with 1 M aqueous HCl (10 mL),
twice with 5% aqueous NaHCO (10 mL) and twice with
2
2
0
0
0
0
(
4H, m, 3 , 5 -CH, 6 -CH -b-CD), 4.94 (1H, s, 1 -CH-b-CD)
2
(
of flight (MALDI-TOF) (m/z): 1490, 1656, 1882, 1988, 2155
Figure 3a); matrix-assisted laser desorption ionization-time
3
brine (10 mL), and then dried over Na SO . The residue was
2
4
+
and 2321 [M+Na] (Figure 3b).
purified by silica gel chromatography eluted with EA/hexane
1
to yield compound 7 as a white solid. Yield: 54%, H NMR
Synthesis of compound 4. A solution of 3 (0.050 g,
.024 mmol) in ACN (5 mL) was added with excess TEA at
0
(300 MHz, CDCl ): d 1.27–1.35 (2H, m, CH CH CH2), 1.42–
3
2
2
ambient temperature and stirred until becoming a completely
clear solution. BA (0.24 mL, 0.242 mmol) was added drop-
wise into the reaction mixture. After overnight stirring, the
reaction mixture was evaporated to remove the solvents.
A 1 M NaOH solution (12 equiv.) was added carefully to the
aqueous solution of compound 4 to exchange excess
n-butylammonium cations with sodium cations. The aqueous
crude was evaporated and dried in vacuum, and then a light
1.49 (11H, s, m, t-Boc, CH CH CO), 1.55–1.59 (2H, m,
2 2
NHCH CH ), 1.61–1.65 (6H, br, CCH CH-ADM), 1.89–1.97
2
2
2
(6H, br, CHCH CH-ADM), 2.03–2.08 (5H, m, CH CHCH -
2
2
2
ADM, CH CH CO) and 3.08–3.14 (2H, m, NHCH CH ).
2
2
2
2
Synthesis of compound 8. Excess TFA (10 mL) was added
ꢀ
to compound 7 (0.75 g, 2.1 mmol) in DCM (30 mL) at 0 C.
The solution was stirred for 2 h, concentrated, washed with
saturated NaHCO3 and brine, and dried over MgSO4.
Compound 8 was obtained as a white solid without further
brown powder, b-CD–MCM–BA (4), was obtained. Yield:
1
1
4
90%, H NMR (300 MHz, 1.1 wt% NaOD in D O): d 0.89–
purification. Yield: 89%, H NMR (300 MHz, 1.2 wt% DCl in
D O): d 1.16–1.26 (2H, m, CH CH CH ), 1.40–1.56 (10H, m,
2
0
1
2
3
.94 (3H, t, CH3CH2), 1.32–1.39 (2H, m, CH3CH2CH2),
.46–1.54 (2H, m, CH2CH2CH2), 1.87 (3H, s, CCH3), 2.22–
.31 (2H, m, CH2CH3CO), 2.49–2.56 (2H, m, CCH2CH2),
2
2
2
2
CH CH CH , CCH CH-ADM), 1.81 (6H, br, CHCH CH-
2
2
2
2
2
ADM), 1.89 (3H, br, CH CHCH -ADM), 2.04–2.09 (2H, t,
2
2
0
0
.18–3.23 (2H, m, CH2CH2NH), 3.54–3.63 (2H, m, 2 , 4 -
0
CH CH CO) and 2.80–2.85 (2H, t, NH CH CH ).
2 2 2 2 2
0
0
CH-b-CD), 3.65–3.92 (4H, m, 3 , 5 -CH, 6 -CH -b-CD), 5.05
Synthesis of compound 9. FITC isomer 1 (0.04 mg,
0.113 mmol) was added in a solution of compound 8
(0.030 g, 0.11 mmol) in MeOH (5 mL), followed by saturated
NaHCO3 (1 mL). After stirring overnight, the reaction
2
0
(
1H, s, 1H, s, 1 -CH-b-CD).
Synthesis of compound 5. b-CD–MCM–CP (5) was
synthesised employing the same preparation method as used

DOI: 10.3109/1061186X.2014.928718
A medusa-like b-CD–MCM 661
mixture was purified by reverse-phase high-performance
liquid chromatography (HPLC) with a linear gradient over
(FLEX02-01D, Correlator.com) and further analysed by a
homemade analysis program coded with LabVIEW 2009
(National Instruments, Austin, TX).
3
flow rate of 6 mL/min. Compound 9 was obtained as an orange
0 min from 50 to 100% of ACN/DIW with 0.1% TFA at a
The fluorescence correlation spectroscopy (FCS) setup
was calibrated to determine the axial and lateral dimensions
of the confocal volume by using a fluorophore, Alexa 488,
1
solid. Yield: 32%, H NMR (300 MHz, MeOD): d 1.40–1.45
(
2H, m, CH CH CH ), 1.61–1.66 (2H, m, CH CH CO), 1.69–
2 2 2 2 2
ꢂ6
2
1
CHCH CH-ADM,
.73 (8H, s, m, CCH CH-ADM, NHCH CH ), 2.01 (9H, br,
2
whose diffusion coefficient is D ¼ 4.35 ꢁ 10 cm /s [31].
2
2
CH CHCH -ADM),
2.13–2.16
2H, t, CH CH CO), 3.65 (2H, br, NHCH CH ), 6.55 (2H, s,
The process followed the procedure described in reference
2
[32]. Calibration showed that the lateral 1/e dimension was
2
2
2
(
2
2
2
2
0
0
2
7
-CH-xanthene), 6.56–6.58 (2H, dd, 4 -CH-xanthene), 7.17–
0
232 nm with the aspect ratio of 7.18, indicating that the
measured focal volume was 0.501 fL.
0
.19 (2H, d, 5 -CH-xanthene), 7.20–7.22 (1H, d, 4 -CH-
0
isobenzofuranone), 7.62–7.64 (1H, d, 5 -CH- isobenzofura-
0
For the measurement of complex formation, a solution of
compound 9 (10 nM) in pH 9.0 phosphate buffer (50 mM) was
prepared with different concentrations of compound 5. Bovine
serum albumin was added to the mixture at the final
concentration of 0.1 mg/mL to prevent the nonspecific
binding between compounds 5 and 9. Each correlation
curve was measured six times for 10 min each (once for
each concentration of compound 5) to suppress the thermal
noises and background signals.
none), 7.82 (1H, s, 7 -CH-isobenzofuranone).
Measurement of pH-sensitive drug release
Release of BA from b-CD–MCM–BA
pH-sensitive release of BA from b-CD–MCM–BA (4) was
1
confirmed by H NMR. b-CD–MCM–BA was dissolved in
1
.2 wt% DCl at the concentration of 3 mg/mL. After incuba-
1
ꢀ
tion with stirring at 37 C for 8 h, H NMR was measured by a
Bruker Avance DPX-300 (Germany) (Figure 4).
Rotating frame overhauser effect spectroscopy
Release of CP from b-CD–MCM–CP
The complex between b-CD and compound 9 and the
complex between b-CD–MCM–CP (compound 5) and com-
pound 9 were prepared by mixing at a 1:1 molar ratio at a
b-CD–MCM–CP (5) was dissolved in a pH 7.4 phosphate
buffer (100 mM) or a pH 5.5 acetate buffer (100 mM) at a
concentration of 3 mg/mL, and each solution was incubated
concentration of 3 mM in a D O-based pH 9.0 phosphate
2
ꢀ
buffer (50 mM). NMR spectra recorded at 25 C on a Agilent
ꢀ
with stirring at 37 C. A YOUNGLIN HPLC (9000 HPLC,
South Korea) equipped with a UV detector and a reverse-
Technologies 400-MR DD2 (Santa Clara, CA) spectrometer.
Rotating frame overhauser effect spectroscopy (ROESY)
experiment was performed using the standard protocols
contained in the spectrometer library (mixing time: 300 ms;
T1 experiment).
phase column (Agilent Eclipse XDB-C18 4.6 ꢁ 150 mm,
5
mm) was used for the HPLC-based measurement. At various
time points, each sample was collected and diluted 10 times
with the same buffer. After filtering through a 0.2-mm
polyvinylidene fluoride syringe filter, the sample was injected
into the HPLC system. A mixture (3:7 v/v) of MeOH:pH 9.0
phosphate buffer (50 mM) was used as the eluent, and the
flow rate was set at 0.5 mL/min. The release of CP was
measured by UV absorbance at wavelengths 245 and 270 nm
Cytotoxicity
Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich)
assay. NIH-3T3 cells (mouse embryonic fibroblast cell
3
line) were seeded at 5.0 ꢁ 10 cells/well in a 96-well plate
(Figure 5).
in 90 mL of Dulbecco’s modified eagle’s medium WelGene
containing 10% foetal bovine serum; WelGene (Daegu,
Complex formation between compound 5 and 9
ꢀ
South Korea) and incubated at 37 C for 24 h. To determine
the cytotoxicity, 10 mL of the solution of each sample with
various concentrations was added into the media with
Fluorescence correlation spectroscopy measurement
A home-built fluorescence confocal microscope setup was
used to measure the autocorrelation of the fluorescent probe,
FITC. A 488-nm continuous blue laser diode (TECBL-20GC-
ꢀ
subsequent incubation at 37 C for 48 h. For the assay, the
cells were washed with Dulbecco’s phosphate-buffered
saline (DPBS; WelGene), followed by the addition of
4
88, World Star Tech, Toronto, Canada) was coupled with a
2
0 mL of filtered MTT solution (2 mg/mL in DPBS). After
single-mode optical fibre (È ¼ 3–5 lm, P1-488-PM-FC,
Thorlabs, Newton, NJ) for the beam clean-up and illuminated
the sample through a water-immersion objective (NA ¼ 1.20,
ꢀ
incubation at 37 C for 2 h, the medium was removed from
the well, and 150 mL of DMSO was added to dissolve the
insoluble formazan particles. The absorbance was measured
at 570 nm using a microplate reader (Molecular Devices Co.,
Menlo Park, CA). The relative cell viability (%) was defined
as the percentage of viable cells compared with the same
percentage in the control (cells treated with DPBS solution).
6
0 ꢁ , f ¼ 3 mm, Olympus, Tokyo, Japan) mounted in a
homemade microscope body. The fluorescence signal was
distinguished with the excitation light by a dichroic mirror
(
ZT488rdc, Chroma, Bellows Falls, VT) and further cleaned
by an emission filter (HQ525/50 m, Chroma). A 1:2 multi-
mode fibre optic coupler ((È ¼ 62.5 lm, FCMM625-50A-FC)
was used as a pinhole to reduce the background from the focal
volume, and two avalanche photodiodes (SPCM-AQR-14-FC,
Perkin Elmer, Waltham, MA) collected the fluorescence
signals. The correlation was measured with a correlator card
Results
Synthesis of b-CD–MCM
We synthesised a pH-sensitive drug carrier based on b-CD
and MCM for efficient drug conjugation, rapid drug release at

6
62 S. Kang et al.
J Drug Target, 2014; 22(7): 658–668
Figure 2. Synthetic schemes of b-CD–MCM and its drug conjugate. (a) Synthesis of b-CD–MCM–BA and b-CD–MCM–CP, (b) Synthesis of
0
1
-methyl-2-(2 -carboxyethyl) maleic anhydride (MCM) and (c) synthesis of FITC-hex-ADM.
acidic pH and simple introduction of functional ligands. The
synthetic scheme is shown in Figure 2(a). After the synthesis
of MCM by the Horner–Wadsworth–Emmons reaction
between triethyl-2-phosphonopropionate and dimethyl-
represent the distribution of various b-CD–(MCM) molecules
accurately, most likely due to the different ionisation degrees
of the b-CD–(MCM) variants.
2
-oxoglutarate (Figure 2b) [29], MCM was coupled with the
Drug conjugation with b-CD–MCM
b-CD molecule via the formation of ester bond between the
carboxyl group of MCM and the hydroxyl group of b-CD. A
b-CD molecule has seven primary hydroxyl groups and
fourteen secondary hydroxyl groups. Among them, primary
At first, a simple primary amine, BA, was used as a model
drug for the optimisation of the conjugation reaction. By
simple mixing in a weakly basic condition, BA was success-
0
1
hydroxyl groups at the 6 -C position of the glucose are more
fully introduced into b-CD–MCM. The H NMR spectrum
reactive to electrophiles, and they preferably react with the
carboxyl group using a carbodiimide-coupling reagent, DIC.
b-CD–MCM was purified from the excess reagents
by dialysis.
shows the formation of the acid amide linkage between BA
and b-CD–MCM (Figure 4). Greater than 90% of MCM
moieties in b-CD–MCM were reacted with BA, and only a
small amount of unreacted BA were remained.
1
The formation of b-CD–MCM was confirmed by H NMR
and MALDI-TOF mass spectrometry (Figure 3). The numbers
Then, CP, an amine-containing antibiotic drug, was
conjugated with b-CD–MCM by a similar method. The
1
of MCM residues per a b-CD–MCM molecule was calculated
to be six by H NMR. In other words, 85% of primary
formation of b-CD–MCM–CP was confirmed by both H
1
NMR and HPLC. The amount of CP with a strong UV
absorption was easily analysed by a UV detector. High
conjugation efficiency (92.5 ± 2.5%) was observed in the
formation of b-CD–MCM–CP. As we applied six equivalents
of CP to b-CD–MCM with six MCM moieties, nearly
exhaustive conjugation was accomplished by the simple
mixing. In addition, the drug content in b-CD–MCM–CP
was calculated to be 28 ± 5.5% by comparing the mass of CP
and the total mass of b-CD–MCM–CP with some residual
sodium and TEA salts.
hydroxyl groups of b-CD were conjugated with MCM on
average. The MALDI-TOF mass spectra exhibited the pres-
ence of b-CD–MCM molecules with various numbers of
MCM residues: b-CD–(MCM)2 (1490), b-CD–(MCM)3
(
1656), b-CD–(MCM)4 (1822), b-CD–(MCM)5 (1988),
b-CD–(MCM)6 (2154) and b-CD–(MCM)7 (2322).
Considering the average conjugation number was six by H
1
NMR and the main peak of the MALDI-TOF spectra was that
of b-CD–(MCM) , the MALDI-TOF spectra could not
4

DOI: 10.3109/1061186X.2014.928718
A medusa-like b-CD–MCM 663
+
electrospray ionisation were also identical (350.1 [M + H]
+
and 699.1 [M + M] , Figure 6b). Based on these data, we
believe that CP was quite stable during the conjugation in
mildly basic conditions and during the release in weakly
acidic conditions.
Complex formation between b-CD–MCM–CP and the
adamantane derivative
To clarify the formation of the complex between the b-CD-
based drug conjugate and an adamantane derivative, we used
FCS, which analyses the variation in the diffusion coefficient
of a fluorophore attached to guest or host [33]. FITC was
conjugated to the adamantane derivative [34] as a probe
(FITC-hex-ADM) (Figure 2c). The average diffusion time of
the fluorophore was measured with varying the concentration
of the host molecule, b-CD–MCM–CP. As the concentration
of b-CD–MCM–CP increased, the average diffusion time also
decreased, confirming the molecular weight increase and the
complex formation (Figure 7). From the graph, the binding
constant between b-CD–MCM–CP and FITC-hex-ADM was
4
ꢂ1
.
calculated to be 1.49 ꢁ 10 M
Furthermore, 2D ROESY NMR experiment was performed
to show the formation of the inclusion complex. Figure 8(a and
b) exhibit the ROESY spectra of the b-CD/FITC-hex-ADM
and b-CD–MCM–CP/FITC-hex-ADM mixtures, respectively.
The a, b and g protons of the adamantane in FITC-hex-ADM
showed strong cross-peaks by interaction with the H3, H5 and
H6 protons in the cavity of native b-CD, and weaker cross-
peaks by interaction with the H2 and H4 protons on the exterior
of the b-CD cavity (Figure 8a). Similarly, the cross-peaks
between the protons of the adamantane derivatives and
b-CD–MCM–CP were also observed (Figure 8b). The spectra
˚
represent the spatial proximity at the 5 A maximal limit among
1
Figure 3. Confirmation of synthesis of b-CD–MCM. (a) H NMR (NMR
solvent: D O) and (b) MALDI-TOF spectra of b-CD–MCM.
the protons of b-CD derivatives and the adamantane moiety.
Both data strongly indicate complex formation between
b-CD–MCM–CP and FITC-hex-ADM.
2
pH-sensitive drug release
Cytotoxicity
The pH-sensitive degradation of the MCM acid amide bond
1
was investigated by H NMR. The methylene protons (marked
The short-term biocompatibility of the b-CD–MCM drug
carrier was preliminarily checked by MTT assay on NIH3T3
cells (Figure 9). Branched polyethylenimine 25 kDa, a well-
known polymeric drug delivery carrier with high cytotoxicity,
was used as a positive control. Up to 100 lM, b-CD, b-CD–
MCM and b-CD–MCM–CP exhibited almost no cytotoxicity
until 48 h.
with x) next to the nitrogen in the intact amide of b-CD–
MCM–BA showed a peak at 3.18–3.23 ppm, while the peak
(
marked with *) was shifted to 2.55–2.62 ppm, equal to the
methylene peak of free BA, in an acidic solution (1.2 wt%
DCl in D2O) (Figure 4). The chemical shift confirmed the
degradation of the MCM acid amide and the release of
conjugated BA.
Discussion
Cumulative release of CP from b-CD–MCM–CP at pH 5.5
and pH 7.4 was measured by HPLC to determine the
degradation kinetics in more detail (Figure 5). At pH 7.4,
less than 20% of CP was released from b-CD–MCM–CP, even
after 5 h. On the other hand, a rapid burst of CP was observed
at pH 5.5, and more than 80% of CP was released within 0.5 h.
The release was almost completed at approximately 95% in
In this study, we intended to develop a pH-sensitive drug
carrier that could sense the small pH drop from 7.4 to 5.5 and
rapidly release the conjugated drug molecules. A backbone
with many conjugating residues was preferred for high drug
contents. In addition, various functions can be introduced by a
simple method for a multi-role drug delivery carrier. We
selected b-CD as the backbone of the carrier due to high
biocompatibility [20], modifiability of many hydroxyl groups
[24] and strong non-covalent complex formation with
adamantane [27]. As a pH-sensitive linker, we selected a
maleic acid amide derivative because it can be easily
synthesised by the simple mixing between the corresponding
1
h. A dramatic difference in release kinetics was observed
between pH 5.5 and pH 7.4. In addition, we confirmed the
stability of CP during the conjugation and release procedure.
1
The H NMR spectrum of the CP released from b-CD–
MCM–CP at pH 5.5 was identical to that of the initial CP
(Figure 6a). The mass spectra of the two CPs obtained by

6
64 S. Kang et al.
J Drug Target, 2014; 22(7): 658–668
Figure 4. Release of BA from b-CD–MCM–
BA at acidic pH measured by H NMR
1
(
2
NMR solvent: D O).
We successfully synthesised b-CD–MCM via a one-step
reaction by the carbodiimide coupling method using DIC
(
Figure 2) under catalysts of DMAP and PTSA. Although
coupling reactions of MCM using thionyl chloride (SOCl ) or
2
oxalyl chloride were reported previously [39], they showed
significant side reactions in the coupling between b-CD and
MCM, so we selected the DIC coupling method to achieve a
high yield of the product. The addition of both DMAP and
PTSA could facilitate the ester formation without the
formation of side products including N-acylurea in polar
solvents [40]. We could control the numbers of MCM
residues that coupled to b-CD by adjusting the amount of
reactants, and consequently obtain a ‘‘medusa-like’’ b-CD–
MCM with six MCM residues on average, which was
1
confirmed by H NMR and mass spectroscopy (Figure 3).
Figure 5. pH-responsive release of CP from b-CD–MCM–CP at pH 5.5
acetate buffer, 100mM; *) and pH 7.4 (phosphate buffer, 100 mM; œ)
measured by HPLC. The error bar represents the standard deviation
n ¼ 3).
Because the face of the secondary hydroxyl groups in b-CD
readily interacts with an adamantane guest molecule [41], the
medusa-like structure by the modification of primary
hydroxyl group would be preferred for the future inclusion
of a functional moiety.
(
(
anhydride and amine and it shows pH-dependent degradabil-
ity. A maleic acid amide derivative has a cis-b-carboxylate
group that can internally attack the carbonyl group of the
amide via five-membered ring formation, and it shows much
higher vulnerability than a simple amide [20,35]. The pH-
sensitivity is also dependent upon the alkyl substituent on the
cis-double bond. Bulkier substituents can accelerate the
internal attack of the b-carboxylate group to increase the
degradability at weakly acidic pH [36]. Among various
maleic acid amide derivatives, we selected the MCM acid
amide because it showed rapid degradability at pH 5.5 owing
to the methyl and carboxyethyl substituents [22,37,38].
We expected a high efficiency in the drug conjugation
with b-CD–MCM because the anhydride group of MCM is
very reactive with nucleophiles such as primary amines in
alkaline conditions, even if the nucleophilic attack is
interfered with by steric hindrance. In both cases of b-CD–
MCM–BA and b-CD–MCM–CP, we similarly observed high
drug efficiency, with the values of 90% and 92.5% for BA and
CP, respectively. By the covalent conjugation with b-CD–
MCM, the drugs can avoid premature release from the drug
delivery carrier in the diluted concentrations in physiological
fluids.

DOI: 10.3109/1061186X.2014.928718
A medusa-like b-CD–MCM 665
Figure 6. Identification of the released CP
1
from b-CD–MCM–CP. (a) H NMR (NMR
solvent: D O) and (b) ESI mass spectra of the
2
released CP.
We investigated the drug-release kinetics of b-CD–MCM–
As mentioned above, b-CD is a cyclic oligosaccharide
composed of seven-membered a-D-glucopyranoside units
linked 1 to 4, and it contains a less hydrophilic interior
space and a more hydrophilic outer surface. This allows b-CD
to form inclusion complexes with various molecules through
host-guest interactions. Adamantane, a cycloalkane consisting
of four connected cyclohexane rings, is the most representa-
tive guest molecule for b-CD. Therefore, we can introduce a
functional moiety such as a targeting ligand or a cell
penetrating peptide into b-CD–MCM by the tethering of an
adamantane residue in the functional moiety and the simple
mixing with b-CD–MCM. For the confirmation of this
concept, we synthesised a FITC-labelled adamantane deriva-
tive to trace the inclusion complex by FCS. The diffusion
CP at pH 5.5 and pH 7.4. Remarkably, CP was dramatically
released from b-CD–MCM at pH 5.5 up to 80% within 0.5 h
and 95% within 1 h, while it was barely released at pH 7.4
(
Figure 5). The chemical stability of CP during the conjuga-
tion and release was also secured. On the basis of the delicate
sensing of the small pH difference and the outstanding release
kinetics at the weakly acidic pH, we believe that b-CD–MCM
can be used as a drug carrier for targeting tumours,
inflammatory tissues or abscesses as well as for early
endosomal escape in intracellular drug delivery. b-CD–
MCM–CP enables the delivery of antibiotics into pneumonia
or other inflamed tissues specifically, to avoid side effects and
overdoses.

6
66 S. Kang et al.
J Drug Target, 2014; 22(7): 658–668
Figure 7. Variation of the average diffusion time measured by FCS to
confirm the complex formation between b-CD–MCM–CP and FITC-
hex-ADM.
coefficient of the fluorophore was decreased significantly due
to the complex formation. The binding constant was
calculated from the ‘‘S’’-type curve between the diffusion
time and the concentration of b-CD–MCM (Figure 7). The
4
ꢂ1
, meaning the
binding constant K was 1.49 ꢁ 10
M
interaction between b-CD–MCM–CP and the adamantane
derivative was similar to the general b-CD/adamantane host–
guest interaction because the general association constant K is
4
ꢂ1
1
–10 ꢁ 10 M [42]. Based on the binding constant, the
inclusion percentage could also be calculated and be shown in
Figure 7. The host–guest inclusion between b-CD–MCM–CP
and adamantane derivative was further confirmed by ROESY
spectra (Figure 8). Strong cross-peaks in b-CD–MCM–CP/
FITC-hex-ADM were comparable with those of b-CD/FITC-
hex-ADM. The medusa-like conjugation of six MCM mol-
ecules in the face of primary hydroxyl groups did not affect
the inclusion significantly. Based on these results, any
functional moieties could be introduced into b-CD–MCM
after the drug conjugation by the formation of the inclusion
complex. Of course, other drug carriers such as polymers or
nanoparticles with adamantane moieties can be easily
modified with b-CD–MCM through the host–guest inter-
action for pH-sensitive conjugation of drugs or other
functionalities.
Finally, we verified that b-CD–MCM and its drug
conjugate showed almost no sign of acute cytotoxicity up to
100 lM in a eukaryotic cell line by MTT assay (Figure 9),
although the long-term toxicity and bioadaptability should be
analysed by various methods in the future.
Figure 8. Expansion of NMR 2D ROESY (NMR solvent: D O) spectra
2
As a novel drug-conjugating system, b-CD–MCM has
great advantages in easy conjugation of drugs and multi-
functionalities as well as rapid release of drugs responding to
small pH drop. CP, an antibiotic model drug in this research,
can be specifically delivered into bacteria-infected sites by a
b-CD–MCM-based drug carrier. b-CD–MCM, with a molar
mass of several thousands, can be used as a pH-sensitive drug
carrier by itself for a site-specific delivery of drugs into
weakly acidic targets such as tumours, abscesses or inflam-
matory tissues. For longer circulation or enhanced perme-
ability and retention effect, b-CD–MCM can be introduced
into other drug delivery carriers with several hundred
nanometres by non-covalent interactions. The future animal
study will be followed for the proof of the specificity and
efficacy of b-CD–MCM-based drug delivery systems.
of the inclusion complex between (a) b-CD and FITC-hex-ADM and (b)
b-CD–MCM–CP and FITC-hex-ADM.
Conclusion
We successfully developed a novel drug delivery carrier,
b-CD–MCM, which has a rapid release profile in response to
a small pH drop. b-CD–MCM possesses several ready-to-use
anhydride residues for simple and almost exhaustive conju-
gation with amine-containing drugs. b-CD–MCM–CP, one of
the drug conjugates, exhibited a remarkable capability to
discriminate between pH 7.4 and 5.5 to release the conjugate
antibiotic. Moreover, the internal cavity of b-CD–MCM–CP
is easily accessible for complexation with other functional
groups with an adamantane residue. Based on these attractive

DOI: 10.3109/1061186X.2014.928718
A medusa-like b-CD–MCM 667
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1
1
1
1
characteristics, b-CD–MCM has great potential as a multi-
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Acknowledgements
This paper is dedicated to my respectable mentor, Prof
Kazunori Kataoka.
2
010;49:2552–5.
Declaration of interest
20. Kirby AJ, Lloyd GJ. Structure and efficiency in intramolecular and
enzymic catalysis: intramolecular general base catalysis. Catalysis
of amide hydrolysis by the carboxy-group of substituted maleamic
acids. J Chem Soc Perkin Trans 2 1976:1753–61.
The authors report no declarations of interest.
This work was supported by the Seoul R&BD program
2
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citraconamic acids, methyl esters, and imides. J Org Chem 1960;25:
(
(
ST110017M0209721); the Basic Science Research Program
NRF-2010-0007118) of the National Research Foundation
1
012–15.
funded by the Ministry of Education, Science and Technology
of Korea; the Mid-Career Researcher Program (NRF-2011-
2
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of a membrane-active agent (EMMA) for cytoplasmic release of
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013569) from the Ministry of Science, ICT and Future
2
2
Planning, Korea; and the GAIA project (2013000550004)
funded by the Ministry of Environment, Korea.
design and synthesis of 6-TBDMS-2,3-methyl beta-cyclodextrin
0
2
-2 thioureido dimer and 6-TBDMS-2,3-methyl (or 2-methyl-3-
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