Self-assembled nanostructures from C60-containing supramolecular complex: Its stimuli-responsive reversible transition and biological antioxidative capacity

Article abstract of DOI:10.1039/c1nj20568d

A thermo-responsive copolymer, P(NIPAM-co-CD), containing β-cyclodextrin as classical host molecule to form highly stable host-guest complexes with 1-adamantane derivatives, was designed and synthesized. The compound tetra-Ad-C₆₀ with a fullerene[60] moiety and four adamantyl heads was designed as a hydrophobic guest molecule. In aqueous solution, P(NIPAM-co-CD) and tetra-Ad-C₆₀ could easily form supramolecular complex by self-assembly of β-cyclodextrin and adamantane, and the supramolecular complex could exist stably as vesicles in ambient environment. Furthermore, the morphology of the supramolecular complex could also be switched reversibly by the change of temperature around the lower critical solution temperature (LCST) of P(NIPAM-co-CD). When temperature was changed around the LCST, vesicles and nano-spheres formed by supramolecular complex could transform reversibly. Due to the presence of the fullerene moieties, the supramolecular complex exhibited excellent ability to scavenge hydroxyl radicals of biological system; furthermore, the antioxidative property could be affected obviously by the change of temperature owing to the thermo-response property of P(NIPAM-co-CD) moiety.

Full text of DOI:10.1039/c1nj20568d

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PAPER
Self-assembled nanostructures from C60-containing supramolecular
complex: its stimuli-responsive reversible transition and biological
antioxidative capacity
Haoyu Wang, Liang Wang, Xiaoguang Wang, Jiayun Xu, Quan Luo and
Junqiu Liu*
Received (in Victoria, Australia) 29th June 2011, Accepted 16th August 2011
DOI: 10.1039/c1nj20568d
A thermo-responsive copolymer, P(NIPAM-co-CD), containing b-cyclodextrin as classical host
molecule to form highly stable host–guest complexes with 1-adamantane derivatives, was designed
and synthesized. The compound tetra-Ad-C60 with a fullerene[60] moiety and four adamantyl heads was
designed as a hydrophobic guest molecule. In aqueous solution, P(NIPAM-co-CD) and tetra-Ad-C60
could easily form supramolecular complex by self-assembly of b-cyclodextrin and adamantane,
and the supramolecular complex could exist stably as vesicles in ambient environment. Furthermore,
the morphology of the supramolecular complex could also be switched reversibly by the change of
temperature around the lower critical solution temperature (LCST) of P(NIPAM-co-CD). When
temperature was changed around the LCST, vesicles and nano-spheres formed by supramolecular
complex could transform reversibly. Due to the presence of the fullerene moieties, the supramolecular
complex exhibited excellent ability to scavenge hydroxyl radicals of biological system; furthermore,
the antioxidative property could be affected obviously by the change of temperature owing to the
thermo-response property of P(NIPAM-co-CD) moiety.
a mass of representative works focus on functionalizing fullerene
with negatively charged carboxylate,8,9 positively charged
Introduction
Fullerene, as the third carbon allotrope,1 has attracted
quaternary ammolonium,10–12 and non-charged functional
groups such as ethylene glycol groups13,14 and polymers,42
increasing attention because of its exciting physical and chemical
properties, which endow fullerene-containing materials with
etc., to enhance the water solubility of fullerene. Another
broad and promising applications in superconductors, ferro-
approach to overcome the insolubility is to encapsulate the
magnets, lubrication, photoconductors, catalysts, and molecular
respective monomer species in a supramolecular structure with
host moiety, such as cyclodextrin,15,16 surfactants,17,18 gels,19
medicines. C60, the most representative member of the full-
or polymers,41 etc. Besides those approaches, fullerene can be
erene family, has been studied in various fields of biomedicine
stabilized in water as nano-scale colloidal assemblies.20–22 For
for its unique properties. As a ‘‘free radical sponge’’, C60 has
demonstrated excellent ability to quench various free radicals
example, nanoscale colloid of C60 can be formed in the
more efficiently than conventional antioxidants.2 Further-
following steps. Firstly, dissolve C60 in water-miscible polar
more, many fullerene-based compounds prepared artificially
organic solvents such as tetrahydrofuran (THF) and mix with
have shown potential practical value, for instance reduction of
injury on ischemic reperfusion of the intestine,3 a decrease in
or dialysis. However, the first two avenues seemed to be more
water; subsequently remove the transfer solvent via distillation
number of cells undergoing apoptosis,4 reduction in free
promising in application of fullerenes in biomaterial fields.
radical levels in organ perfusate,5 and neuroprotective effects.6
Because of the extreme hydrophobicity of C60, most water-
soluble derivatives of C60 are amphiphilic,39,40 while amphiphilic
Though, in recent years, people have had much more interest
in studying potential bioactive water-soluble fullerene deriva-
molecular organization is one of the widely used methods in
tives, emphasizing their capacity to quench free radicals, the
supramolecular chemistry to construct nano-architectures.23
extreme hydrophobicity7 is a serious barricade for the broad
This method uses the balanced hydrophilic-hydrophobic structural
prospects on bio-medical research and application. Therefore,
motifs to give rise to micelles, bilayers and further vesicular
structures, and its advantage makes it possible to solubilize the
State Key Laboratory of Supramolecular Structure and Materials,
College of Chemistry, Jilin University, Changchun 130012, People’s
Republic of China. E-mail: junqiuliu@jlu.edu.cn;
Fax: +86-431-85193421
extremely hydrophobic C60 in polar media and to utilize the
propensity of amphiphilic fullerenes to form prominent
assemblies.10,24,49
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b-Cyclodextrin, composed of 7 D-(+)-glucose units linked by
a-1,4-linkages, has been deeply introduced into supramolecules
for its unique structure and easy availability.25,26 b-Cyclodextrin
has a good solubility in aqueous solution, while its geometry
makes a hydrophobic inner cavity and various organic, inorganic
and biological molecules can be trapped in the cavity to form
supramolecular inclusion complexes. These complexes have
induced a lot of significant developments in supramolecular
systems for biomedical and pharmaceutical applications.25
Stimuli-responsive polymers have attracted much attention
for their remarkable changes in physical and chemical properties
with small changes of external stimuli; therefore, they have
potential in a variety of applications in biomedical fields.27,28
Among them, temperature- and pH-responsive mechanisms
are more interesting and have been extensively investigated28
due to the relative convenience and efficient stimuli. Poly
(N-isopropylacrylamide) (PNIPAM), a representative thermo-
responsive polymer, has been widely studied in supramolecular
chemistry29–31 for its phase transition at its lower critical
solution temperature (LCST) of 32 1C.32,33 Besides PNIPAM,
many similar polymers can undergo the same phase transition
around their LCSTs.34,35 Some copolymers based on
PNIPAM can alter their morphologies in aqueous solution
when temperature changes around their LCSTs.36–38,43 Therefore,
PNIPAM also has a significant value in targeted drug delivery.44
In this work, we applied a thermo-responsive copolymer
consisting of host sites that could recognize guest molecules to
conjugate insoluble derivatives of C60, in contrast to conventional
water-soluble C60, for the purpose of realizing its solubility. The
fabrication of thermo-responsive assemblies was based on the
complex consisting of thermo-responsive P(NIPAM-co-CD)
and a derivative of fullerene with adamantyl moiety. The
complex can self-assemble to vesicles for the amphiphilic
structure. It is noteworthy that the morphology of assemblies
could be changed reversibly from vesicles to nano-spheres
when temperature changed around the LCST of P(NIPAM-
co-CD). Furthermore, the assemblies have an excellent ability
to scavenge hydroxyl radicals of a biological system due to the
introduction of the derivative of fullerene into the complex,
and the scavenging efficiency could be obviously influenced by
the change of temperature owing to the thermo-responsive
property of the supramolecular polymer.
Acryloyl-b-cyclodextrin was synthesized according to Harada
et al.’s report.45
Instrumentation
The characterizations of the compounds were performed with a
VARIAN 300 (300 MHz) 1H and 13C NMR spectrometer.
Scanning electron microscopy (SEM) observations were carried
out on a JEOL FESEM 6700F scanning electron microscope
with primary electron energy of 3 kV. Transmission electron
microscopy (TEM) observations were carried out on a JEOL
JEM 3010 TEM. The sizes of the assemblies were determined by
the Malven Zetasizer NanoSeries instrument. UV-Vis spectra
were obtained using a Shimadzu 3100 UV-Vis-NIR spectro-
photometer. Data was acquired and analyzed by using ultraviolet
spectroscopy software. The temperature for UV-Vis absorption
studies was controlled within ꢀ0.5 1C by use of a LAUDA
compact low-temperature thermostat RC6 CP. The buffer
pH values were determined with a METTLER TOLEDO
320 pH meter.
Synthesis of compound 1
A solution of adamantane-1-carboxylic acid (2 g, 11 mmol)
and thionyl chloride (3 g, 25.2 mmol) in dry THF (30 mL) was
heated to 78 1C and stirred for 3 h. After solution had been
evaporated, the pale crude was redissolved in dry THF
(10 mL) and dropped into the solution of tetraethylene glycol
(6.4 g, 33 mmol) and triethylamine (3.3 g, 33 mmol) in ice bath.
After stirring for 5 h, the precipitate was filtered and washed
with THF (3 ꢁ 10 mL), and combined organic fractions were
evaporated. The crude product was purified by silica gel
chromatography, eluting with ethyl acetate to yield 1 as a
colourless oil (1.38 g 35%). 1H NMR (300 MHz, CDCl3):
d 3.6–4.3 (m, 16H, –O–CH2–), 2.01 (s, 3H, adamantane),
1.90 (s, 6H, adamantane), 1.72 (m, 6H, adamantane).
Synthesis of compound 2
Dicyclohexylcarbodiimide (DCC) (578 mg, 2.8 mmol) and a
catalytic amount of 4-dimethylamiopryidine (DMAP) were
added to a stirred solution of 1 (1 g, 2.8 mmol) and malonic
acid (146 mg, 1.4 mmol) in 50 ml dry tetrahydrofuran at room
temperature under nitrogen. After overnight reaction, the
product precipitate was removed by filtration, and the filtrate
was evaporated. The crude product was purified by silica gel
chromatography, eluting with ethyl acetate and petroleum
ether (gradient) to yield 2 as a colourless oil (0.93 g, 85%).
Experimental section
1H NMR (300 MHz, CDCl3):
d 3.64–4.28 (m, 16H,
Materials
–O–CH2–), 3.43 (s, 2H, H), 1.99 (broad s, 3H, adamantane),
1.87 (broad s, 6H, adamantane), 1.69 (m, 6H, adamantane).
MALDI-MS: calcd 780, found 779.
Toluene, tetrahydrofuran, dimethyl formamide and chloroform
were purchased from Beijing Chemical Plant and were purified in
appropriate methods. N-Isopropylacrylamide was acquired from
Aldrich and purified by recrystallization from a 3 : 2 toluene/
hexane mixture. a,a0-Azoisobutyronitrile was obtained from
Aldrich and purified by recrystallization from methanol. Tetra-
ethylene glycol and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
were from J&K Chemical Ltd. and used without further
purification. Fullerene[60] (C60) was purchased from Yongxin
Technology Ltd. for direct use. Adamantane-1-carboxylic acid
was from Alfa and was used directly. Thiobarbituric acid, iodine
and rhodamine B (Rh–B) were from Shenyang Guoyao Co., Ltd.
Synthesis of compound 3
1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (39.3 mg, 0.26 mmol)
in 1 mL of toluene was added to a stirred solution of 2 (70 mg,
0.09 mmol), C60 (29.7 mg, 0.0414 mmol), and I2 (22.8 mg,
0.09 mmol) in 100 mL of toluene, and the mixture was stirred
for 24 h under a nitrogen atmosphere. The reaction mixture was
loaded on top of a short silica gel chromatography column and
eluted with toluene to remove the unreacted fullerene. Further
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elution with toluene/ethanol (50 : 1) afforded 3 as a dark red solid
1
pH 7.4, and maintained at 0 1C. The concentration of the
mitochondria protein was determined by coomassie brilliant
blue47 using bovine serum albumin as the standard.
(30.2 mg, 32%). H NMR (300 MHz, CDCl3): d 4.61 (m, 8 H),
4.20 (m, 8 H), 3.90–3.64 (m, 48 H), 2.00 (broad s, 12 H adamantane),
1.89 (broad s , 24 H adamantane), 1.70 (m, 24 H adamantane).
13C NMR (75 MHz, CDCl3) 177.4, 163.2, 147.1, 146.8, 146.4, 145.5,
145.4, 145.2, 145.0, 144.5, 144.3, 143.3, 142.4, 141.5, 140.2, 139.1,
139.1, 139.1, 138.5, 138.4, 77.6, 77.5, 69.1, 68.7, 68.6, 66.1, 66.0, 63.1,
40.6, 38.7, 36.4, 27.8. MALDI-MS: calcd 2276, found 2277.
Mitochondrial swelling measurements. The swelling of mito-
chondria was characterized by turbidity at 520 nm according
to the method reported by Hunter et al.48 Change of mito-
chondrial morphology results from the damage of mitochondrial
membrane, and the absorbance decreases with the increasing
of the swelling.
Synthesis of compound 4
Polymerization was conducted in a 50 mL round-bottomed
flask equipped with stirring bar. According to the recipes as
follows, in general, 190 mg (1.68 mmol) of NIPAM and 100 mg
(0.0841 mmol) of acyloyl-b-cyclodextrin were dissolved in
20 mL of DMF and heated to 65 1C under a nitrogen purge
for 30 min at least to remove dissolved oxygen. A solution of
a,a0-azoisobutyronitrile (AIBN) initiator (2.9 mg AIBN in 0.6 mL
DMF) was then injected, and the polymerization was allowed
to proceed for 24 h under continuous nitrogen bubbling and
stirring. The resulting copolymer was decanted and allowed to
cool. The product was purified by dialysis against water using
semipermeable membranes with cutoff molar masses of
14 000 g molꢂ1 for 36 h. P(NIPAM-co-CD) fraction with
relatively narrow polydispersity was collected and then lyophilized.
The molar ratio of [NIPAM]/[CD] determined from 1H NMR
was 35. GPC analysis of P(NIPAM-co-CD) revealed a Mn
77 210 and a polydispersity Mw/Mn 1.31.
Evaluation of the lipid peroxidation
Thiobarbituric acid (TBA) was used as a marker for lipid
peroxidation, and fullerene derivatives as antioxidants in
ferrous sulfate/ascorbate-induced mitochondrial damage to
determine the levels of lipid peroxidation. The procedure
was as follow in brief: 1mL liquid reactant was mixed with
1mL trichloroacetic acid (TAC, 100 g Lꢂ1) and 1mL thio-
barbituric acid (TBA, 5 g Lꢂ1), and the mixture was incubated
at 85 1C for 30 min. After cooled, the mixture was centrifuged
with 3000 rpm for 10 min and determined the absorbance of
the supernatant at 532 nm.
Results and discussion
Thermo-response of P(NIPAM-co-CD)
It is well-known that PNIPAM possesses good water-solubility
in cold and dilute aqueous solution but poor above 32 1C, due
to its phase transition behavior above its LCST. Thereupon
the copolymer P(NIPAM-co-CD) we synthesized, with a
measured amount of cyclodextrin, should naturally have the
thermo-responsive ability. P(NIPAM-co-CD) was directly soluble
in distilled water at 25 1C. Heated above 36 1C, an opalescence
Preparation of the P(NIPAM-co-CD)(tetra-Ad-C60)
Tetra-Ad-C60 (3.6 mg) was dissolved in 500 mL THF, and the
solution was slowly injected into a 10 mL of P(NIPAM-
co-CD) in water (4 mg mLꢂ1) under ultrasonic at ambient
temperature. Yellow brown opalescence appeared immediately,
which indicated the formation of assemblies. THF was
removed by dialysis for 36 h, and the fine dispersion was kept
at room temperature in dark place over one week at least for
further study.
characteristic micellar solution appeared. Fig.
1 shows
temperature-dependent optical transmittance obtained for
5 mg mLꢂ1 aqueous solution of P(NIPAM-co-CD). It can
be clearly seen that the transmittance starts to decrease
dramatically at 34–35 1C, indicating that the copolymer
is becoming hydrophobic above its LCST. The SEM
(see Fig. 3A) and dynamic light scattering (DLS) (see
Fig. 2a) results can confirm the formation of the P(NIPAM-
co-CD) assemblies. The average diameter of the particles
obtained from DLS is 200 nm.
Hydroxyl radical (HOꢃ) scavenging activity
Interception of hydroxyl radicals by each sample was deter-
mined by the UV absorption of Rh–B in Fenton reaction. The
assay was based on the competition between the Rh–B and the
samples of assemblies for HOꢃ. Hydroxyl radicals were
generated by the classical Fenton reaction with the reaction
mixture containing freshly prepared 11.2 mM Rh–B, 500 mM
FeSO4 (5 mmol stock solution, freshly made), PBS (50 mmol
stock solution, pH 7.0, EDTA 5 mmol) and 20 mmol H2O2
with or without the complex we prepared. Agents were
injected into sample cell in sequence as following: Rh–B,
FeSO4, PBS (containing EDTA), sample (with or without),
ultrapure water, H2O2. The molar concentration of
P(NIPAM-co-CD)(tetra-Ad-C60) is defined by tetra-Ad-C60’s.
Preparation and characterization of supramolecular complex
A derivative (tetra-Ad-C60) of C60 with adamantyl moiety and
thermo-sensitive polymer P(NIPAM-co-CD) with cyclodextrins
have been synthesized as the routes mentioned above
(Scheme 1). Owing to the hydrophobic host–guest interaction
between b-cyclodextrin and adamantane, tetra-Ad-C60
and P(NIPAM-co-CD) could easily assemble in aqueous
environment, although a small amount of THF existed in
the system. For the good water-solubility of P(NIPAM-
co-CD) and the hydrophobicity of C60 moiety, a secondarily
self-assembly of the supramolecular complex was carried out
in lots of water after adamantyl moiety was encapsulated into
the cavity of b-cyclodextrin. After dialysis, THF was removed
and the solution was stable in ambient environment.
Mitochondrial swelling measurements
Mitochondria preparations. Bovine heart mitochondria were
isolated from fresh bovine heart according to the method of
previous researchers,46 and suspended in 0.25 mol Lꢂ1 sucrose,
10 mmol Lꢂ1 EDTA and 25 mmol Lꢂ1 HEPES-NaOH buffer,
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Fig. 1 Temperature-dependent optical transmittance at 600 nm
obtained for 10 mg mLꢂ1 aqueous solution of P(NIPAM-co-CD).
Fig. 3 SEM and TEM micrographys of assemblies. (A) SEM of
P(NIPAM-co-CD) sample forming vesicles at 40 1C; (B) SEM of
P(NIPAM-co-CD)(tetra-Ad-C60) sample forming vesicles at 20 1C; (C)
SEM of P(NIPAM-co-CD)(tetra-Ad-C60) sample forming aggregates at
40 1C; (D) TEM of P(NIPAM-co-CD)(tetra-Ad-C60) sample forming
vesicles at 20 1C; (E) TEM of P(NIPAM-co-CD)(tetra-Ad-C60) sample
forming aggregates at 40 1C. TEM of P(NIPAM-co-CD)(tetra-Ad-C60)
sample forming aggregates at 20 1C returned from 40 1C.
The heating rate is 1 1C minꢂ1
.
Fig. 2 (a) The size distribution of P(NIPAM-co-CD) was determined
via dynamic light scattering at 20 1C and 40 1C respectively; (b) the size
distribution of P(NIPAM-co-CD)(tetra-Ad-C60) was determined via
dynamic light scattering at 20 1C and 40 1C respectively.
The solution of the supramolecular complex appeared light
yellow due to the C60 moiety. The opalescence of the solution
was not very obvious under the LCST of P(NIPAM-co-CD) but
apparent above the temperature. And as the temperature fell below
the LCST, the solution could resume its original appearance. DLS
was employed to measure the size and size distributions of the
assemblies of supramolecular complex (see Fig. 2b). At ambient
temperature (20 1C), the assemblies, which are composed of
P(NIPAM-co-CD) and tetra-Ad-C60, have an average diameter
of 160 nm and a polydispersity index (PDI) of 0.57 estimated from
the relative line width, and it is obviously different from the size of
P(NIPAM-co-CD) alone. The SEM and TEM images correspond
with the results of DLS. From the SEM image (see Fig. 3B), it can
be observed that the assemblies of the supramolecular complex are
hollow vesicles and the TEM image (see Fig. 3D) confirms that the
shells of the vesicles consist of copolymer outer layer and tetra-
Ad-C60 inner layer. In addition, from another point of view, the
results of DLS, SEM and TEM at lower temperature could prove
the formation of supramolecular complex.
Scheme 1 Synthetic procedures for tetra-Ad-C60 and P(NIPAM-co-CD).
the hollow vesicles transformed into nano-spheres that could be
confirmed by SEM and TEM images (see Fig. 3C and E). From
the TEM image (see Fig. 3E), there was not an apparent layer
in these nano-spheres in contrast with assemblies at ambient
temperature. Therefore, we considered that the arrangement of
the complex was disordered in the assemblies at higher temperature
(above LCST), as shown in the illustration (see Scheme 2). In
addition, the reversibility of the transformation of P(NIPAM-co-
CD)(tetra-Ad-C60) was tested. Fig. 4 shows that the transformation
of the complex is completely reversible after multiple heating-
cooling cycles in accordance with the TEM image (see Fig. 3F).
Hydroxyl radical (HOꢃ) scavenging activity in Fenton reaction
When temperature increased above the LCST, the average
diameter of the assemblies decreased to 103 nm (see Fig. 2b), and
We confirmed the hydroxyl radical scavenging activity of
P(NIPAM-co-CD)(tetra-Ad-C60) by Fenton reaction, and the
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results also echo previous researches from others.50–52 Because
fullerene molecules can efficiently quench fluorescence of
Rh–B in solution and that had been confirmed in our
pre-assays, the interception of hydroxyl radicals in Fenton
reaction of each sample was determined by the UV-Vis
absorption of Rh–B instead of fluorescent excitation and
emission spectrum. Rh–B has a strong absorption at 554 nm
in aqueous solution and the absorption will reduce, when it is
oxidized by oxidizers, accompanied by its molecular structure
broken. In this work, Rh–B was oxidized by hydroxyl radicals
that were generated by Fenton reagent. The presence of
complex P(NIPAM-co-CD)(tetra-Ad-C60) could efficiently
inhibit the oxidation of Rh–B according to the results from
Fig. 5, and the performance of protection depends on the
concentration of P(NIPAM-co-CD)(tetra-Ad-C60). Therefore,
it could be proved indirectly that P(NIPAM-co-CD)(tetra-
Ad-C60) can effectively scavenge hydroxyl radicals.
Fig. 4 Changes of the size distribution of P(NIPAM-co-CD)(tetra-
Ad-C60) during heating-cooling cycles between 20 1C to 40 1C.
Because the copolymer P(NIPAM-co-CD) is thermo-
responsive and the morphology of assemblies of P(NIPAM-
co-CD)(tetra-Ad-C60) can be changed by temperature, we
investigated the hydroxyl radicals scavenging extent of the
complex at a fixed concentration and different temperatures to
explore how the changes of temperature and morphology
influence scavenging property. The variable temperature
Fenton reaction was carried out in the presence of
P(NIPAM-co-CD)(tetra-Ad-C60) at a concentration of 0.45 mmol
from 26 to 40 1C. It obviously shows us (Fig. 6) that the
scavenging efficiency of hydroxyl radicals is relative stable
when temperature is below LCST and reduced when tempera-
ture is above LCST. The decrease of efficiency may be
attributed to the change of morphology of the assemblies
around LCST, since all the conditions were constant except
temperature. According to Scheme 2, the complex self-
assembled as vesicles when it was below LCST, and the outer
layers were hydrophilic, so that the inner C60 segments might
contact with hydroxyl radicals relatively easily. While it was
above LCST, the contact between hydroxyl radicals and C60
may be blocked to a certain extent, because the vesicles
transformed into hydrophobic nano-spheres and the C60
Fig. 5 UV-Vis absorption spectra of Rh–B in Fenton reaction: (a)
control, Rh–B + PBS + Fe(II); (b) Rh–B + PBS + Fe(II) + H2O2 +
0.54 mM P(NIPAM-co-CD)(tetra-Ad-C60); (c) Rh–B + PBS + Fe(II)
+ H2O2 + 0.36 mM P(NIPAM-co-CD)(tetra-Ad-C60); (d) Rh–B +
PBS + Fe(II) + H2O2 + 0.18 mM P(NIPAM-co-CD)(tetra-Ad-C60);
(e) Rh–B + PBS + Fe(II) + H2O2.
segments spread uniformly. Therefore, the scavenging
efficiency of hydroxyl radicals decreases when temperature is
above LCST, and lowers as temperature rises.
Mitochondrial swelling measurements
Hydroxyl radical, as one of the known strongest oxidants, is of
very short lifetime and high reactivity, and it is extremely
harmful to organisms as it can react with almost all cells.
Mitochondria, as the most important organelle in the eukaryotic
cell, is the main site of aerobic respiration, and is the principal
Fig. 6 Scavenging efficiency of hydroxyl radical of P(NIPAM-co-
CD)(tetra-Ad-C60) in Fenton reaction by UV-Vis from 26 to 40 1C at
concentration of 0.54 mM.
Scheme 2 Schematic illustration of the change in structure of the
assemblies by temperature.
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generated MDA accounts for 91.2%, 77.9%, and 51.5% of
the damage, namely 8.8%, 22.1%, and 48.5% of MDA are
inhibited respectively.
Conclusions
In this study, we have designed and synthesized adamantyl
fullerene as the guest molecule and copolymer P(NIPAM-co-
CD) as the host molecule to construct a supramolecular
complex. For the thermo-responsive ability of P(NIPAM-co-
CD), the morphology of assemblies formed by the supra-
molecular complex can alter reversibly from vesicles to
nano-spheres by change of temperature in aqueous solution.
Due to the C60 moiety, the complex shows a remarkable ability
for hydroxyl radical scavenging and could inhibit biological
oxidative damage on mitochondria efficiently in vitro. Further-
more, the change in morphology could obviously influence the
property of hydroxyl radicals scavenging of the supramolecular
complex. This method bodes well not only for designing novel
water-soluble fullerenes but also for constructing drug delivery
systems with bioactivities.
Fig. 7 Effect of P(NIPAM-co-CD)(tetra-Ad-C60) on the swelling of
mitochondria. (&) Control; (J) damage; (b) damage + 0.36 mM
P(NIPAM-co-CD)(tetra-Ad-C60); (K)damage
P(NIPAM-co-CD)(tetra-Ad-C60); (m) damage
P(NIPAM-co-CD)(tetra-Ad-C60).
+
0.54 mM
0.9 mM
+
source of ROS (reactive oxygen species) in organisms, and the
oxidative damage of mitochondria can represent the severity
of some diseases.
Therefore, we introduce P(NIPAM-co-CD)(tetra-Ad-C60)
into the assay of oxidative damage of mitochondria to
evaluate its hydroxyl radical scavenging property. As shown
in Fig. 7, the swelling of mitochondria was obvious after the
addition of Fe2+/Vc, and it was apparently inhibited in the
presence of P(NIPAM-co-CD)(tetra-Ad-C60). We investigated
the inhibition to swelling in different concentrations of
complex, and the results showed that the inhibition effect would
be more obvious with an increase of concentration of complex.
Acknowledgements
Financial support from the Natural Science Foundation of
China (No: 91027023, 20874036, 20921003, 21004028), the
NSFC for Outstanding Younger Scientist (No. 20725415),
the National Basic Research Program (2007CB808006) and
Key Laboratory of Rare Earth Chemistry and Physics, Chang-
chun Institute of Applied Chemistry (RERU2011008)
Evaluation of the lipid peroxidation
Notes and references
As a characterized product, malondialdehyde (MDA) can be
employed to characterize the lipid peroxidation. In damage
groups, the content of MDA gradually accumulated by time,
while the content reduced after radical scavenger was added
into the system. As shown in Fig. 8, supramolecular complex
P(NIPAM-co-CD)(tetra-Ad-C60) can apparently protect mito-
chondria from oxidative damage, and the extent of protection
is proportional to the concentration. When the concentration
of P(NIPAM-co-CD)(tetra-Ad-C60) is 0.36 mmol, 0.54 mmol,
and 0.9 mmol, after it reacts for 50 min, the content of
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