PH responsive smart carrier of [60] fullerene with 6-amino-cyclodextrin inclusion complex for photodynamic therapy

Article abstract of DOI:10.1039/c2jm34791a

A new pH responsive smart carrier of C₆₀ was prepared with 6-amino-γ-cyclodextrin (ACD) for photodynamic therapy. C₆₀ was rapidly released from its inclusion complex with ACD at pH 6.7, due to electrostatic repulsion between ammonium

Full text of DOI:10.1039/c2jm34791a

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PAPER
pH responsive smart carrier of [60] fullerene with 6-amino-cyclodextrin
inclusion complex for photodynamic therapy†
d
e
d
abcd
Kazuyuki Nobusawa,* Motofusa Akiyama, Atsushi Ikeda and Masanobu Naito*
Received 20th July 2012, Accepted 18th September 2012
DOI: 10.1039/c2jm34791a
A new pH responsive smart carrier of C60 was prepared with 6-amino-g-cyclodextrin (ACD) for
photodynamic therapy. C60 was rapidly released from its inclusion complex with ACD at pH 6.7, due to
electrostatic repulsion between ammonium groups, followed by a rapid C60 release at slightly acidic
cancer cell surfaces.
Photodynamic therapy (PDT) can be regarded as a patient-
friendly anticancer therapy because it does not involve ionizing
radiation, which allows it to be used repeatedly without cumu-
lative long-term complications since it does not appear to target
DNA. Recent studies on PDT with first generation photosensi-
tizers have shown some efficacy in the treatment of a variety of
malignant and premalignant conditions, including head- and
stability and controlled release of the g-CD/C60 inclusion
complex considering external stimuli, such as pH, temperature,
and light, is highly desired for practical photodynamic therapy
applications. Recently, several approaches targeting the extra-
cellular pH of cancer cells have been developed using water
soluble polymers with covalently attached hydrophobic anti-
22,23
cancer drugs as side chains.
Here a slightly acidic extracel-
1
2,3
4
neck-cancers, lung-cancer, mesothelioma, Barrett’s esoph-
lular pH (pH ¼ 6.4–7.2) was utilized as an external stimulus for
chemical cleavage of the covalent linkage between the polymer
backbone and the anticancer moieties. However, these methods
require dexterous molecular designs to provide adequate chem-
ical responsiveness at specific pH conditions. Therefore, a smart
carrier that rapidly releases C60 in response to the extracellular
pH of cancer cells remains challenging. In this study, we
demonstrated for the first time that a cyclodextrin (CD) deriva-
tive bearing primary amino groups at the primary face acts as a
smart carrier of non-substituted hydrophobic C60 to the slightly
acidic cancer cells. Consequently, the efficient uptake of C60
exhibited excellent photodynamic activity against cancer cells.
As a pH responsive moiety, a primary amine was monofacially
5,6
7
8,9
agus, prostate and brain tumors. Among the various types
of first generation photosensitizers, [60] fullerene (C60) is an
emerging material for photodynamic therapy because photoex-
cited C60 generates activated oxygen species, resulting in efficient
10–15
extinction of the vicinal tissues.
However, for realistic
applications its insolubility in water is limiting. To overcome this
shortcoming, many attempts at improving the water solubility of
1
6,17
C
60 have been developed with a variety of chemical
and
18–21
physical modifications.
In this context, inclusion complexes
of C60 with g-cylodextrin (g-CD) have received a lot of attention
as a facile solubilization technique, without elaborate chemical
treatments. However, from a practical viewpoint of the smart
delivery of C60 to the specific cancer cell, extremely stable
hydrophobic interactions between C60 and g-CD inhibits uptake
into cancer cells. Therefore, a rational design leading to optimal
24,25
introduced into the 6-positions of CD (ACD) (Fig. 1a).
Here
we expected that the amino groups at the 6-positions would not
interfere in the inclusion of C60 with ACD under neutral pH
conditions because C60 is only accessible from the bottom face of
ACD to form the inclusion complex (ACD/C ). Only when the
6
0
a
TU–NIMS Joint Research Center, School of Materials Science and
Engineering, Tianjin University, 92 Weijin Road, Nankai District,
amino groups of ACD were protonated did the upper face
expand due to electrostatic repulsion between the protonated
amino groups, followed by shrinkage of the bottom face. In
conjunction with the deformation of the ACD rings, the included
Tianjin 300072, P.R. China
b
National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki
3
05-044, Japan. E-mail: NAITO.Masanobu@nims.go.jp; Tel: +81 029
8
60 4783
C60 is rapidly squeezed from the inclusion complex to the vicinal
c
JST PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho,
Kawaguchi, Saitama 332-0012, Japan
cancer cells (Fig. 1b and c). In this study, ACD/C60 was prepared
by a solid-state mechanochemical reaction as described elsewhere
d
Graduate School of Materials Science, Nara Institute of Science and
Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. E-mail:
2
6
(
see ESI†). Briefly, pristine C60 was vigorously shaken with
n-kazuyu@ms.naist.jp; Fax: +81 743 72 6018; Tel: +81 743 72 6017
e
ACD powder by high-speed vibration milling at 1800 rpm for 20
min. The resulting mixture was dispersed in water, and then the
insoluble portion was removed by centrifugation. The obtained
supernatants were kept as a stock solution. The ACD/C60 solu-
tion was colored light-violet, and did not exhibit precipitation
Department of Applied Chemistry, Faculty of Science and Engineering,
Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 113-8551, Japan
†
Electronic supplementary information (ESI) available: Synthetic
procedures, Experimental details, DLS, UV-vis absorption
measurement data. See DOI: 10.1039/c2jm34791a
2
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Fig. 1 (a) Chemical structure of 6-amino-g-cyclodextrin (ACD), and (b)
schematic representation of 2 : 1 inclusion complex of ACD and C60. (c)
Proposed mechanism of release from the smart carrier of C60 with ACD
inclusion complex in response to the protonation of amino groups at
extracellular cancer cell pH (pH 6.4–7.2).
over a week under neutral pH conditions. This suggests that C60
is homogeneously isolated in ACD/C60 under neutral pH
conditions. The stoichiometric ratio of the host–guest complex
could not be directly estimated by conventional titration because
high-speed vibration milling is a solid-state reaction process.
However, from the evidence from UV-vis (ultraviolet-visible)
Fig. 2 Photographs of (a) ACD/C60 and (b) g-CD/C60 at pH ranges of 4
to 9. (c) Changes in UV-vis spectra of ACD/C60 upon changing the pH
from 4.2 to 8.9. (d) Changes in UV-vis absorption of ACD/C60 at 450 nm
(red) and amount of 1 N HCl (blue) required to adjust the final pH from
4.2 to 8.9.
27
absorption data and a recent molecular simulation study, we
proposed that an isolated ACD/C60 complex forms a 2 : 1
complex (Fig. 1). By changing the pH from 9 to 4, the color of the
ACD/C60 solution rapidly changed from light-violet to brownish
yellow at a critical pH of 6.7, indicating that C60 formed a
colloidal aggregation (Fig. 2a). From dynamic light scattering
analysis, the average diameter of the C60 colloidal aggregation
was estimated to be ca. 20 nm (Fig. S1, ESI†). Here the C60
colloidal aggregations should be stabilized by the protonated
ACD under lower pH conditions. On the other hand, the light-
violet solution of g-CD/C60 remained unchanged in the pH
region between 4 and 9, due to the absence of the pH responsive
moiety in g-CD over this pH region (Fig. 2b and S2, ESI†). The
significant changes in the solution color were further elucidated
by UV-vis spectroscopy. Thus, the ACD/C60 solution exhibited
an intense UV signal at 332 nm, originating from isolated C60
molecular mechanics calculations were performed using PCFF
force fields (Materials Studio 4.0, Accelrys Inc., San Diego, CA)
(
Fig. 3). To simplify the calculations, a 1 : 1 inclusion complex
was adapted as a model for an initial configuration, instead of a
: 1 inclusion complex. Non-, half-, and fully-protonated ACDs
2
were used as transition models for ACD gradually becoming
28
(
Fig. 2c). Upon changing the pH from 8.9 to 4.2, this peak
became broad and red-shifted to 338 nm. In addition, a broad
signal around 450 nm appeared. Similar spectral changes are
29
often seen in the aggregation of C60
.
Unlike the g-CD/C60 inclusion complex, ACD/C60 exhibited a
unique release of C60 in response to subtle pH changes slightly
lower than neutral conditions. We performed a pH titration of
ACD/C60 with 1 N HCl. The pH of ACD/C60 gradually changed
without a sudden step upon the addition of 1 N HCl (Fig. 2d,
blue solid line). Therefore, the amino groups of ACD appear to
adopt multistep protonation, probably due to steric effects
among neighboring amino groups. In spite of the gradual
changes in the protonation of amino groups, C60 was rapidly
released at a critical pH of 6.7 (Fig. 2d, red solid line). To address
the unique release behavior, computational studies with
Fig. 3 Proposed structures of ACD/C60 complexes. (a) Top view of C60
and non-protonated ACD, (b) C60 and half-protonated ACD, and (c) C60
0
0
0
and fully-protonated ACD. (a ), (b ), and (c ) represent the side views of
the corresponding models. The atom colors gray, white, red, blue and
+
orange represent C, H, O, N and N respectively. To simplify the
calculation, 1 : 1 inclusion complexes of C60 and ACD were employed.
PCFF was used as a force field (Materials Studio 4.0).
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J. Mater. Chem., 2012, 22, 22610–22613 | 22611

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protonated. In the half-protonated ACD, amino groups are
alternately protonated. Consequently, non-protonated ACD
could tightly accommodate C60 in ACD, due to hydrophobic
interactions between C60 and the inner cavity of ACD (Fig. 3a
Indeed, g-CD/C60 did not express photodynamic activity at
either pH 6.4 or 7.4 (Fig. 4b), and exhibited a lack of dark
toxicity under the same pH conditions (Fig. 4a). On the other
hand, ACD/C60 showed excellent photodynamic activity. Thus,
cell viability significantly decreased with an increase of ACD/C60
concentration from 2 to 10 mM (Fig. 4d). Similar to g-CD/C60, an
apparent dark toxicity was not evident (Fig. 4c). Furthermore,
the cell viability at pH 6.4 was greater than that at pH 7.4.
Judging from the pH titration of ACD/C60, the protonated ACD
readily released C60 at pH 6.4. Following that, the released C60
could be taken up into the cancer cells within 30 min. Here, C60 is
thought to be adsorbed to the hydrophobic parts of the cell
0
and a ). Interestingly, half-protonated ACD did not show
significant changes in conformation, and C60 remained stably
0
located in the ACD (Fig. 3b and b ). This is because hydrophobic
interactions between C60 and ACD overcame the deformation
energy derived from electrostatic repulsion between protonated
amino groups. Eventually, when the amino groups were fully
protonated, the upper face of ACD increased from 0.9 nm in
diameter to 1.5 nm. Conversely, the bottom face of ACD
0
14,15,18,20,21,33,34
decreased from 1.1 nm in diameter to 0.9 nm (Fig. 3c and c ).
membrane, as is often seen in artificial membranes.
Responding to the deformation, C60 was immediately squeezed
from the inner cavity of the ACD, followed by the formation of
the colloidal aggregations.
Thus, active oxygen species were produced by photoexcited C60
incorporated into the cancer cell membrane, and then unsatu-
rated phospholipids or membrane proteins were oxidized,
leading to efficient cell death. Here it is noteworthy that cell
viability at pH 7.4 upon visible light irradiation was significantly
decreased down to 30%, although this effect is less than that at
pH 6.4. It is well known that the extracellular pH of the human
cancer cell is acidified at approximately pH 5–6. Thus, even
under the neutral buffer condition (pH 7.4), the extracellular pH
vicinal HeLa cell appears to be rather acidic. Therefore, ACD/
C60 is thought to release C60 nearby the HeLa cell surface, fol-
lowed by uptake of a certain amount of C60 into the cell surface
(Fig. S3, ESI†). Consequently, significant photodynamic activity
was expressed even at pH 7.4. On the other hand, in the case of
pH 6.4, the ACD/C60 forms colloidal aggregations surrounded
by protonated ACDs with an average diameter of 20 nm in the
bulk solution. Therefore, a large amount of C60 aggregations in
the bulk solution were taken up into the HeLa cells, probably
through endocytosis, followed by the expression of the more
efficient photodynamic activity (Fig. 4). To further confirm the
proposed uptake mechanism of C , the direct observation of C
Finally, we demonstrated the in vitro photodynamic activity of
the ACD/C60 complex using human cervical cancer HeLa cells
(
see ESI†). ACD/C60 solution was added to the cell cultures.
Here, the solutions were adjusted to be pH 7.4 and pH 6.4 with
PBS buffer, corresponding to the extracellular pH of normal cells
and cancer cells, respectively. Successively, the cell cultures were
exposed to visible light (400–500 nm) for 30 min. After contin-
uous light irradiation, cell viability was evaluated using a WST-8
assay (Cell Counting Kit-8: Dojindo Laboratories Co., Kuma-
moto, Japan). As a reference, g-CD/C60 was used as a non-
responsive C60 carrier at the extracellular pH of cancer cells. It
has been reported that the photodynamic activity of g-CD/C60 is
30
negligibly low, although it does not show dark toxicity. This is
because C60 could not be released from the stable g-CD/C60
,
1,32
3
leading to an inhibition of C60 uptake into the cancer cells.
6
0
60
in HeLa cells was demonstrated with TEM (transmission elec-
tron microscopy) (see ESI†). Consequently, after 30 min incu-
bation of ACD/C60 in the presence of HeLa cells at pH 6.4, it was
revealed that C60 existed both in the cell membrane (Fig. 5a) and
within the intracellular vehicle (Fig. 5b). These results strongly
supported the conclusions that (i) direct insertion to the cell
membrane and (ii) uptake into the cell through endocytosis were
simultaneously carried out (Fig. 6).
Fig. 5 TEM images of ACD/C60 localization in a HeLa cell (a) without
0
Fig. 4 Cell viability of HeLa cells treated with g-CD/C60 ((a) in the dark;
b) after photoirradiation) and ACD/C60 ((c) in the dark; (d) after pho-
toirradiation). The viability was tested at pH 7.4 (blue columns) and pH
.4 (red columns). *p < 0.05 as compared with the activities at pH 7.4.
staining and (b) stained with uranyl acetate/lead citrate. (a ) Enlarged
0
(
image of the square in (a). (b ) Enlarged image of the square in (b). C60
was located in the cell membranes (indicated by arrows in (a)) and within
the intracellular vehicle (the square in (b)).
6
2
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C
60 aggregation surrounded by protonated ACDs.
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C60 to cancer cells, resulting in significant photodynamic activity
under slightly acidic conditions, as a mimic of extracellular
cancer cell conditions. The rational design of a sensitive pH
responsive carrier was achieved by adjusting the stability of the
ACD/C60 inclusion complex. In this system, the protonation of
the amine groups acted as a trigger for deformation of the ACD
rings, leading to the efficient release of C60 in response to subtle
changes in pH around neutral. We also succeeded in demon-
strating in vitro photodynamic activity of ACD/C60. C60 was
rapidly taken up into the cancer cells at pH 6.4, resulting in
significant photodynamic activity under the pH conditions of
extracellular cancer cells. The present simple but effective pH
responsive smart carrier with amino-cyclodextrin could be
applicable for a variety of drug deliveries in physiological envi-
ronments by optimizing the ring size of ACD and/or the amine
derivatives with different basicity.
1
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This research was partially supported by PRESTO/JST, the
Green Photonics Project at NAIST sponsored by MEXT. K.N.
thanks Iketani Science and Technology Foundation for financial
support. The authors are grateful to Ms. Nagai at NAIST for
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1 To evaluate the amount of g-CD/C60 taken up into HeLa cells under
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J. Mater. Chem., 2012, 22, 22610–22613 | 22613