A novel pH-sensitive (±)-α-tocopherol-5-fluorouracil adduct with antioxidant and anticancer properties

Article abstract of DOI:10.1039/c1cc13821a

A novel pH-sensitive (±)-α-tocopherol-5-fluorouracil (VE-5-FU) adduct with antioxidant and anticancer properties for antioxidant-based cancer chemoprevention was synthesized and utilized for selective drug release in the stomach.

Full text of DOI:10.1039/c1cc13821a

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COMMUNICATION
A novel pH-sensitive (Æ)-a-tocopherol–5-fluorouracil adduct with
antioxidant and anticancer propertiesw
a
a
a
a
a
a
Dong-Wei Li, Fang-Fang Tian, Yu-Shu Ge, Xin-Liang Ding, Jia-Han Li, Zi-Qiang Xu,
a
a
a
a
ab
Mei-Fang Zhang, Xiao-Le Han, Ran Li, Feng-Lei Jiang and Yi Liu*
Received 27th June 2011, Accepted 24th August 2011
DOI: 10.1039/c1cc13821a
A novel pH-sensitive (Æ)-a-tocopherol–5-fluorouracil (VE-5-FU)
adduct with antioxidant and anticancer properties for antioxidant-
based cancer chemoprevention was synthesized and utilized for
selective drug release in the stomach.
urgent problem. Thus, the application of a natural molecule as
the drug carrier would be a promising strategy to solve this
problem.
a-Tocopherol, the most active component of natural vitamin E,
consists of a chromanol head, which is located at the water–
lipid interface, and a long saturated phytyl chain tail, which is
Cancer, aging, atherosclerosis, and some other serious diseases
have been confirmed to correlate with low density lipoprotein
8
buried within the hydrocarbon chains in the lipid bilayers.
(
LDL), cell membranes, and DNA exposed to oxidative
The structure of a-tocopherol affords its ease of incorporation
into membranes, which might facilitate the transport of conjugates
1
stress. It is generally believed that the formation of growth
9
into the interior of cancer cells. The antioxidant activity of
promoting oxidants (reactive oxygen species, ROS) is a major
2
‘
‘catalyst’’ of the tumor promotion and progression stages.
a-tocopherol relies on its effectiveness in donating hydrogen
from the hydroxyl group of the chromanol ring to reactive
10
radicals, so when a-tocopherol exists in the form of an ester
ROS involve a series of oxidants, such as hydrogen peroxide
1
), singlet oxygen ( O
ꢀ
), free radicals (DPPH ), etc.,
(
H
2
O
2
2
among which the radicals attract much attention because they
3
can drive carcinogenesis by damaging DNA and proteins.
conjugate the antioxidant activity will be hidden (as shown in
Scheme 1). Based on these properties, we report herein (to the
best of our knowledge) the first pH-sensitive (Æ)-a-tocopherol–
5-fluorouracil conjugate (VE-5-FU) as a dual-acting drug
(antioxidant–anticancer) for antioxidant-based cancer chemo-
prevention, which can hold the conjugate form at a higher pH
value (pH 7.0–8.0) and release the two single units at a lower
pH value (pH 1.0–3.0). This dual-acting adduct could have
potential application for delivering the bifunctional drug to the
target site of lower pH value, such as the stomach. Additionally,
the systematic biophysical investigation of the interaction
between serum albumin (SA) and VE-5-FU is also discussed,
as the absorption, distribution, and metabolism of drugs are
strongly dependent on the interactions between SA and drugs.
Thus, some works are devoted to investigating the radical-
scavenging properties of the presented anticancer drugs to
4
enlarge the application of these drugs as antioxidants. However,
the antioxidant–anticancer hybrid as a dual-acting drug has
been seldom reported up to now.
Recently, stimuli-responsive drugs have attracted more and
more attention for controlling the release rate and targeting
the release site, because the release can be triggered by
5
environmental temperature, pH and/or light. Among those
drugs, pH-responsive drugs have been paid much attention
because of large variations in physiological pH at different
6
body sites under normal as well as pathological conditions.
For example, the pH value of the human body increases from
the stomach (pH 1.0–3.0) to the small intestine (pH 6.5–7.0),
7
and to the colon (pH 7.0–8.0). The most common strategy for
facilitating the pH-responsive controllable release is the use of
5b,c
synthetic polymer–drug conjugates. One significant limitation
of this strategy, however, is the use of an exogenous polymer,
metabolism and excretion of which in the body is still an
a
State Key Laboratory of Virology & Key Laboratory of Analytical
Chemistry for Biology and Medicine (MOE), College of Chemistry
and Molecular Sciences, Wuhan University, Wuhan 430072,
P. R. China. E-mail: yiliuchem@whu.edu.cn; Fax: +86-27-6854067;
Tel: +86-27-68756667
Department of Chemistry and Life Sciences, Xianning University,
Xianning 437005, P. R. China
b
Scheme 1 Schematic representation of the VE-5-FU acid-triggered
drug release process. The 5-FUA (blue) and Vitamin E (red) are
highlighted in the scheme.
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc13821a
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10713–10715 10713

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Scheme 2 The synthesis route for VE-5-FU. (a) ClCH
refluxed, 2 h; (b) (Æ)-a-tocopherol, DCC/THF, Ar, 50 1C, stirred,
8 h.
2
COOH, KOH,
4
The coupling of the two segments to obtain the conjugate
VE-5-FU was done using general ester synthesis techniques
Scheme 2), and the radical-scavenging properties of VE-5-FU
(
were tested in a semi-aqueous system using 2,2-diphenyl-1-
Fig. 2 In vitro cell assays of 5-FUA and VE-5-FU against HeLa cells.
Cells incubated with drugs at different concentrations (2.6–330 mM) at
37 1C for 48 h.
ꢀ
picrylhydrazyl (DPPH ) as a radical species (for details and
characterization see ESIw). Generally, when VE-5-FU was
added to a deaerated EtOH–H O (1 : 1) solution of DPPH
ꢀ
2
at different pH (pH 1.0–8.0), the visible absorption band at
ꢀ
25 nm due to DPPH disappeared gradually, indicating that
cell line was monitored by the MTT assay. The results of cell
viability of VE-5-FU and its precursor 5-FUA are shown in
Fig. 2. Based on the MTT results, the enhancement of
cytotoxicity for VE-5-FU compared to 5-FUA indicated
that the cell membrane-permeable a-tocopherol moiety might
facilitate the transport of conjugates into the interior of cancer
cells. Moreover, the acidic intracellular compartments such as
endosomes (pH 5.0–6.0) and lysosomes (pH 4.0–5.0) might
also trigger the release of VE-5-FU.
5
ꢀ
the DPPH -scavenging reaction of VE-5-FU was taking place.
ꢀ
In those cases in which pH = 1.0, the DPPH radical was
unstable, so the measurement of the scavenging property at
pH = 1.0 was imprecise. As illustrated in Fig. 1a, the radical-
scavenging properties of VE-5-FU were time- and pH-dependent.
ꢀ
Under the neutral conditions (pH = 7.0–8.0), the DPPH
capture behavior of VE-5-FU was relatively weak, with the
ꢀ
majority (490%) of DPPH remaining for 5 h. However,
DPPH -scavenging was much enhanced at pH of 2.0, and
ꢀ
It is widely accepted in the pharmaceutical industry that
an understanding of the chemistry of the various classes
of pharmaceutical interactions with SA can suggest new
ꢀ
over 55% of the initial DPPH was scavenged within 5 h, due
to highly efficient hydrolysis of the acid-sensitive VE-5-FU
ester conjugate. To optimize the reaction conditions, radical-
scavenging properties with different VE-5-FU concentrations
are also discussed, as shown in Fig. 1b, which indicated that
we had the chance to optimize our drug system by using higher
VE-5-FU concentrations.
1
1
approaches to drug therapy and design. We first studied
the influence of VE-5-FU on the binding affinity and secondary
structure of SA. The Stern–Volmer quenching constants (KSV
4
)
À1
for VE-5-FU–SA and 5-FUA–SA systems were 1.223 Â 10 M
4
À1
and 1.492 Â 10
M
, respectively (see Fig. S3 in ESIw).
Obviously, there were no significant changes in KSV for
VE-5-FU compared to its precursor 5-FUA, which illustrated
that the derivatization of the traditional anticancer drug 5-FUA
had almost no influence on the binding affinity of the drug–SA
system. As shown in CD spectra (see Fig. S4 in ESIw), the
contents of a-helix, b-sheet and unordered calculated by
SELCON3 suggested that the binding of VE-5-FU to SA
had no significant impact on the secondary structure of SA, and
therefore, VE-5-FU could be successfully carried by SA without
detriment to the function of SA.
To assess the anticancer activity of VE-5-FU and 5-FUA
directly, the cell viability of the human cervical cancer HeLa
A
We then investigated the affinity constant (K ) and binding
site of the VE-5-FU–SA system via experimental and theoretical
methods. The results of UV-vis absorption spectra (see Fig. S5
in ESIw) confirmed that the probable binding mechanism of
VE-5-FU to SA was a complex formation process, rather than
a collision process. Thus, we could use the electrochemical
impedance spectroscopy (EIS) experiment to obtain K for
A
1
2
the VE-5-FU–SA complex. Comparing the EIS of a bare
Au electrode with that of a SA-modified Au electrode, Rct
(
the diameter of the semicircle of the Nyquist plot) increased
strikingly after deposition of SA (Fig. 3a). This should be
attributed to the blocking of electron transport after deposition
of SA on the Au surface (Fig. 3b). Fig. 3c clearly shows that
ꢀ
Fig. 1 (a) The DPPH -scavenging properties of VE-5-FU in semi-
R
ct increased with increasing concentrations of VE-5-FU
aqueous solutions at rt at pH 2, 3, 5, 7, 8. The concentrations are
solutions. According to the Langmuir adsorption isotherm:
ꢀ
5
0 mM for DPPH , and 100 mM for VE-5-FU, respectively. (b) The
effect of VE-5-FU concentration on DPPH -scavenging properties.
ꢀ
max 0 A 0 A
Rct = (Rct) C K /(1 + C K )
1
0714 Chem. Commun., 2011, 47, 10713–10715
This journal is c The Royal Society of Chemistry 2011

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the score (stands for Àlog K
d
) calculated between site I and
VE-5-FU is much higher than that of site II, which was
consistent with the site marker competitive result. The long
hydrophobic chain tail of VE-5-FU was inserted into a
hydrophobic cave created by the residues Leu242, Phe246,
Ala314, Ile313, Ile287, Ala284, Leu283, Val264, Leu257,
Leu261, and Val258 (Fig. 4b). One hydrogen bond had been
observed in the VE-5-FU–SA system: O4 in the VE-5-FU
molecule formed one hydrogen bond with the H atom in
Arg218 (see Fig. S7 in ESIw). These results suggested that
the main forces of VE-5-FU–SA interaction were hydrophobic
interaction and van der Waals force, and the hydrogen bond
also stabilized the formation of the VE-5-FU–SA complex.
In conclusion, we report herein a novel pH-sensitive (Æ)-a-
tocopherol–5-fluorouracil (VE-5-FU) adduct with antioxidant
and anticancer properties. This hybrid could be released in an
acidic medium rather than in a neutral medium. The adduct also
possesses enhanced anticancer activity against HeLa cells and
moderate binding affinity to serum albumin. This dual-acting
adduct could have potential application for delivering the
bifunctional drug for antioxidant-based cancer chemoprevention
to the target site of lower pH value, such as the stomach.
This work was supported by the financial support of the
National Natural Science Foundation of China (21077081,
Fig. 3 (a) Nyquist plots of impedance spectra recorded for the Au
electrode before and after SA deposition. (b) AFM image (recorded in the
tapping mode) of the Au surface after deposition of SA. Structures with
lateral dimensions in the range of 41–57 nm and a height of up to 8 nm
are seen. These are not compatible with dimensions of single SA obtained
from the X-ray crystallography (18.3 nm  9.5 nm for the in-plane
dimensions and 3.8 nm for the height of SA). These structures indicate
the formation of aggregates, possibly due to nonspecific protein binding.
(c) Nyquist plots for impedance measurements corresponding to the SA
modified Au electrode with different VE-5-FU concentrations. Dotted
symbols represent experimental data; solid lines show the curves fitted to
the equivalent circuit (the inset) with best parameters. (d) Linear plot of
the Langmuir isotherm of the data presented in (c).
20921062), Program for Changjiang Scholars and Innovative
Research Team in University (IRT1030), and the Fundamental
Research Funds for the Central Universities (201120302020013).
a plot of C
0 0
/Rct as a function of C yielded a straight line from
5
À1
Notes and references
which the affinity constant K
A
= 2.47 Â 10 M was deduced
as shown in Fig. 3d. It showed that the binding constant
between VE-5-FU and SA was moderate, and VE-5-FU could
be stored and carried by this protein in the body.
1
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According to Sudlow’s nomenclature, two primary sites
2
(
I and II) have been identified for ligands binding to SA.
The identification of the binding site of VE-5-FU in SA was
studied by two different methods. The first method was a site
marker competitive experiment, which was carried out by using
warfarin and ibuprofen as site marker fluorescence probes
for monitoring sites I and II of SA, respectively. The results of
the site marker competitive experiment indicated that the
binding site of VE-5-FU was mainly located within site I of
SA (see Fig. S6 in ESIw). A second method, Surflex-Dock
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1
3
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Fig. 4 Docking results of the VE-5-FU–SA system. (a) Binding site
of VE-5-FU in SA. VE-5-FU is shown in a space-filling model.
9
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This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10713–10715 10715