C60 fullerene as promising therapeutic agent for correcting and preventing skeletal muscle fatigue

Abstract

Background
Bioactive soluble carbon nanostructures, such as the C60 fullerene can bond with up to six electrons, thus serving by a powerful scavenger of reactive oxygen species similarly to many natural antioxidants, widely used to decrease the muscle fatigue effects. The aim of the study is to define action of the pristine C60 fullerene aqueous colloid solution (C60FAS), on the post-fatigue recovering of m. triceps surae in anaesthetized rats.

Results
During fatigue development, we observed decrease in the muscle effort level before C60FAS administration. After the application of C60FAS, a slower effort decrease, followed by the prolonged retention of a certain level, was recorded. An analysis of the metabolic process changes accompanying muscle fatigue showed an increase in the oxidative stress markers H2O2 (hydrogen peroxide) and TBARS (thiobarbituric acid reactive substances) in relation to the intact muscles. After C60FAS administration, the TBARS content and H2O2 level were decreased. The endogenous antioxidant system demonstrated a similar effect because the GSH (reduced glutathione) in the muscles and the CAT (catalase) enzyme activity were increased during fatigue.

Conclusions
C60FAS leads to reduction in the recovery time of the muscle contraction force and to increase in the time of active muscle functioning before appearance of steady fatigue effects. Therefore, it is possible that C60FAS affects the prooxidant-antioxidant muscle tissue homeostasis, subsequently increasing muscle endurance.

Full text

Prylutskyy et al. J Nanobiotechnol (2017) 15:8
DOI 10.1186/s12951-016-0246-1
RESEARCH
C60 fullerene aspromising therapeutic
agent forcorrecting andpreventing skeletal
muscle fatigue
Yurij I. Prylutskyy1, Inna V. Vereshchaka2, Andriy V. Maznychenko2*, Nataliya V. Bulgakova2, Olga O. Gonchar3,
Olena A. Kyzyma1,4, Uwe Ritter5, Peter Scharff5, Tomasz Tomiak6, Dmytro M. Nozdrenko1, Iryna V. Mishchenko7
and Alexander I. Kostyukov2
Abstract
Background: Bioactive soluble carbon nanostructures, such as the C60 fullerene can bond with up to six electrons,
thus serving by a powerful scavenger of reactive oxygen species similarly to many natural antioxidants, widely used
to decrease the muscle fatigue effects. The aim of the study is to define action of the pristine C60 fullerene aqueous
colloid solution (C60FAS), on the post-fatigue recovering of m. triceps surae in anaesthetized rats.
Results: During fatigue development, we observed decrease in the muscle effort level before C60FAS administration.
After the application of C60FAS, a slower effort decrease, followed by the prolonged retention of a certain level, was
recorded. An analysis of the metabolic process changes accompanying muscle fatigue showed an increase in the
oxidative stress markers H2O2 (hydrogen peroxide) and TBARS (thiobarbituric acid reactive substances) in relation to
the intact muscles. After C60FAS administration, the TBARS content and H2O2 level were decreased. The endogenous
antioxidant system demonstrated a similar effect because the GSH (reduced glutathione) in the muscles and the CAT
(catalase) enzyme activity were increased during fatigue.
Conclusions: C60FAS leads to reduction in the recovery time of the muscle contraction force and to increase in the
time of active muscle functioning before appearance of steady fatigue effects. Therefore, it is possible that C60FAS
affects the prooxidant-antioxidant muscle tissue homeostasis, subsequently increasing muscle endurance.
Keywords: C60 fullerene, Skeletal muscles fatigue, Electrical stimulation, Oxidative stress markers, Antioxidant system
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
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and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
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Background
Skeletal muscle fatigue is the defence mechanism against
overload and leads to the development of painful muscle
sensitivity [1–3]. Muscle fatigue develops after physi-
cal activities of varying intensities and often leads to
acute pain, which can then lead to various chronic dis-
ease states [4, 5]. Muscle fatigue is a result of the prod-
ucts of incomplete oxygen oxidation, such as reactive
oxygen species (ROS), including peroxides, free radicals,
and oxygen ions [6]. During the course of lipid peroxi-
dation, unsaturated fatty acids are formed from various
fatty acid derivatives and metabolites, such as malondi-
aldehyde and hydroperoxide fatty acid [7]. e excessive
accumulation of ROS (oxidative stress) can lead to signif-
icant functional changes due to damage to different cell
components [8]. An example is the lipid peroxidation of
biological membranes, which promotes the disruption of
their structure and increases their permeability [9]. Cell
protection against such damage is provided by the anti-
oxidant system. Mach etal. [10] used pycnogenol as an
antioxidant, and its use is accompanied by an increase
in the levels of both oxidized and reduced NAD+ in the
serum, as well as increased muscle strength. In studies
of muscle fatigue, endogenous antioxidants, such as an
N-acetylcysteine [11] and β-alanine [12], are widely used
and speed up the muscle recovery process after fatigue.
Open Access
Journal of Nanobiotechnology
*Correspondence: maznychenko@biph.kiev.ua
2 Department of Movement Physiology, Bogomoletz Institute
of Physiology, Bogomoletz Str. 4, Kiev 01024, Ukraine
Full list of author information is available at the end of the article
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Prylutskyy et al. J Nanobiotechnol (2017) 15:8
In this context, bioactive soluble carbon nanostruc-
tures, such as the pristine C60 fullerenes, may be con-
sidered potential antioxidants [13]. C60 fullerene easily
bonds with up to six electrons, can serve as a powerful
scavenger of ROS [13–15], and is superior to the major-
ity of natural antioxidants, including vitamins C and E
and carotenoids, in regard to its antioxidant capacity.
As a result, it prevents oxidative stress dissemination in
thymocytes [16] and shows a protective effect following
the ischemia–reperfusion injury of skeletal muscle [17].
Additionally, water-soluble pristine C60 fullerenes can
penetrate through the plasma membrane of cells [18, 19].
erefore, the use of C60 fullerenes may have a powerful
antioxidant effect on the contractile apparatus of striated
muscle, thereby facilitating its functional recovery after
experimentally induced fatigue.
e aim of this study was to investigate the effect of
water-soluble pristine C60 fullerenes on the recovery
dynamics of the contractile properties of rat m. triceps
surae (TS) after the development muscle fatigue under
conditions of long-term activation.
Methods
Material preparation andcharacterization
A highly stable reproducible pristine C60 fullerene aque-
ous colloid solution (C60FAS) at a concentration of
0.15mg/ml was prepared according to a previous pro-
tocol [20, 21]. Briefly, for the preparation of C60FAS
we used a saturated solution of pure C60 fullerene
(purity >99.99%) in toluene with a C60 molecule con-
centration corresponding to maximum solubility near
2.9 mg/ml, and the same amount of distilled water in
an open beaker. e two phases formed were treated in
ultrasonic bath. e procedure was continued until the
toluene had completely evaporated and the water phase
became yellow colored. Filtration of the aqueous solu-
tion allowed to separate the product from undissolved
C60 fullerenes. e pore size of the filter during the filtra-
tion of the aqueous solution was smaller than 2µm (Typ
Whatmann 602 h1/2). e purity of prepared C60FAS
(i.e., the presence/absence of any residual impurities, for
example carbon black, toluene phase) was determined by
HPLC and GC/MS analysis. e maximal concentration
of C60 fullerenes in water 0.15mg/ml was obtained by
this method.
e state of C60 fullerenes in aqueous solution was
monitored using atomic force microscopy (AFM). Under
AFM analysis, the sample was deposited onto a cleaved
mica substrate (V-1 Grade, SPI Supplies) by precipitation
from an aqueous solution droplet. Sample visualization
was performed in semi-contact (tapping) mode (Fig.1a,
b). AFM measurements were performed after the com-
plete evaporation of the solvent.
Small-angle neutron scattering (SANS) measurements
(Fig.1c) were carried out at the YuMO small-angle dif-
fractometer at the IBR-2 pulsed reactor (JINR, Dubna,
Russia) in the time-of-flight mode with the two-detector
setup [22]. Treatment of the raw data was performed by
the SAS program [23].
Procedure andexperimental groups
Male Wistar rats, weighing 280–350g, were used in the
study. e use of the animals was approved by the Ethics
Committee of the Institute and performed in accordance
with the European Communities Council Directive of 24
November 1986 (86/609/EEC).
e animals were divided into 4 groups. In the experi-
ments, the m. triceps surae fatigue was induced by elec-
trical stimulation of n. tibialis. Saline solution (group 1,
n=6) or C60FAS (F-injection) 0.1–0.15mg/kg (group 2,
n=6) was administered into the left TS after the devel-
opment of fatigue. en, fatigue of the right TS was
induced. e data obtained from the ipsilateral (left)
side were considered to be the control values vs. those
obtained from the contralateral side. e dose range of
0.1–0.15 mg/kg C60FAS does not present any acute or
subacute toxicity in rats [13]. e rats of group 3 (n=6;
animals with fatigue of both TS without any injections)
and group 4 (n= 6; intact animals) were used only for
biochemical studies. After the experiment, the TS of
all animals in all groups were removed for biochemical
analysis.
It is important to note that a dose of 0.1–0.15mg/kg
C60FAS applied in our experiments does not present any
acute or subacute toxicity in animals: it was significantly
lower than the maximum tolerated dose of C60 fullerene,
which was found to be 5g/kg both for oral or intraperito-
neal administration to rats [13]. No toxic effects or death
have been fixed under the action of C60 fullerenes after
their oral administration to rats in total dosage of 2g/kg
for 14days [24]. Finally, it was shown [13] that water-sol-
uble C60 fullerenes administered intraperitoneally to rats
(0.5mg/kg) were subjected to clearance from the organ-
ism within 2–4days.
e animals in groups 1 and 2 were anaesthetized
with ketamine (100mg/kg “Pfizer”, USA) combined with
xylazine (10 mg/kg, “Interchemie”, Holland), tracheos-
tomized and artificially ventilated (out of necessity). e
left and right TS muscles were separated from the sur-
rounding tissue, and their tendons were detached at the
distal insertions. e n. tibialis was separated from the
tissue and cut proximally, and all branches of the nerve,
except those innervating the TS, were cut. is nerve was
mounted on a bipolar platinum wire electrode for elec-
trical stimulation. e hindlimb muscles and nerves were
covered with paraffin oil in a pool formed from skin flaps.
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Prylutskyy et al. J Nanobiotechnol (2017) 15:8
e TS muscle was connected via the Achilles tendon to
the servo-control muscle puller. e muscle tension was
measured by semi-conductor strain gauge resistors glued
on a stiff steel beam mounted on the moving part of a lin-
ear motor.
To induce muscle fatigue, 1–3 (30min duration) series
intermittent high-frequency electrical stimulation was
used (Fig.2a), separated by rest intervals of 10–20min.
Each series consisted of trains of 0.2-ms rectangular
pulses at a rate of 40/s at 12.4s duration and separated
by 5s intervals of rest (Fig.2b). e stimulus current was
set to 1.3–1.4 times the motor threshold. Note, if mus-
cle fatigue developed in less than 30 min, stimulation
was interrupted (it was predicted that fatigue develop-
ment occurred when there was a muscle force decrease
of more than 50% of the initial data). After the end of the
12.4-s-stimulation, the muscle was stretched, and the
change in length had a bell-shaped form (one period of
4 Hz sinusoidal signal with corresponding phase lock-
ing) of 3.5mm amplitude and 2s duration (Fig.2b; bot-
tom row). e muscle reaction to the stretches appeared
as a tension increase after continuous stimulation. ese
stretches were applied before the post-stimulation
twitches to remove, or at least diminish, the after-effects
remaining from the continuous stimulation [1]. e sig-
nals (stimulus pulses, muscle tension and other) were
sampled via DAC-ADC device (CED Power 1401).
Biochemical experiment
For biochemical analysis, the excised m. triceps surae
(soleus and gastrocnemius) were rapidly dissected, free of
fat and tendon, divided into several portions and stored
in liquid N2. For reduced glutathione (GSH) analysis, tis-
sue samples were transferred into a medium containing
1 N perchloric acid (1:10 w/v) and homogenized with
a motor-driven Potter–Elvehjem glass homogenizer.
e resultant homogenate was centrifuged at 10,000g
for 10 min (4 °C). e GSH content was spectropho-
tometrically measured [25]. For the enzyme activity
assays and H2O2 and lipid peroxidation assays, the mus-
cle samples were thawed and homogenized in 50 mM
a
b
c
Fig. 1 AFM images (tapping mode) of C60 fullerene particles on the
mica surface, which were precipitated from C60FAS with an initial
concentration of 0.15 mg/ml (a, b). Arrows indicate the height of
the individual particles. Experimental SANS curve (points) for C60FAS
(0.15 mg/ml). Solid lines correspond to the model curve obtained by
the IFT procedure. Insert: the pair distance distribution function as a
result of the IFT procedure for scattering from C60 fullerene nanoparti-
cles present in the C60FAS (c)
◂
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Prylutskyy et al. J Nanobiotechnol (2017) 15:8
phosphate buffer with 2mM EDTA (pH 7.4) at 4°C (1:9
w/v). Homogenates were then centrifuged for 15min at
15,000g (4°C), and the post mitochondrial supernatant
was stored at −70°C.
Oxidative damage in the tissue was measured using the
thiobarbituric acid reactive substances (TBARS) assay.
TBARS were isolated by boiling tissue homogenates for
15min at 100°C with thiobarbituric acid reagent (0.5%
2-thiobarbituric acid/10% trichloroacetic acid/0.63 M/
dm3 hydrochloric acid) and measuring the absorbance at
532nm. e results are expressed as nM TBARS/mg pro-
tein, using ɛ=1.56×105 dm3/M1/cm1 [26].
e H2O2 concentration in the tissue homogenates was
measured using the FOX method, which is based on the
peroxide-mediated oxidation of Fe2+, followed by the
reaction of Fe3+ with xylenol orange (o-cresolsulphoneph-
thalein 3′,3″-bis[methylimino] diacetic acid, sodium salt).
is method is extremely sensitive and is used to meas-
ure low levels of water-soluble hydroperoxide present in
the aqueous phase. To determine the H2O2 concentra-
tion, 500μl of the incubation medium was added to 500μl
of assay reagent (500 μM ammonium ferrous sulphate,
50mM H2SO4, 200μM xylenol orange, and 200mM sorb-
itol). e absorbance of the Fe3+-xylenol orange complex
(A560) was detected after 45min. Standard curves of H2O2
were obtained for each independent experiment by add-
ing variable amounts of H2O2 to 500μl of basal medium
mixed with 500μl of assay reagent. Data were normalized
and expressed as μM H2O2 per mg protein [27].
Catalase activity was measured by the decomposition
of hydrogen peroxide, determined by a decrease in the
absorbance at 240nm [28].
GSH was determined using Ellman’s reagent. One mil-
lilitre of supernatant was treated with 0.5ml of Ellman’s
reagent (5.5′-dithio-bis-nitrobenzoic acid in abs. ethanol)
and 0.4M Tris HCl buffer with 2mM EDTA, pH 8.9. e
absorbance was read at 412nm in a spectrophotometer
[25].
e protein concentration was estimated using the
method of Bradford with bovine serum albumin as a
standard. All chemicals were purchased from Sigma,
Fluka and Merck and were of the highest purity.
Data analysis
In the electrophysiological part of the study, each stimula-
tion series (30min) was divided into three equal portions
(Fig.2a), which were averaged (maximum 33 stimulation
in one portion). e average value of the first portion was
set to 100%, and the other series were normalized in rela-
tion to this (for each hindlimb). e peak amplitudes of
the front (P1) and rear of the front (P2) (maxima ampli-
tudes at the site, duration of 1s, Fig. 2b) of the muscle
strength of each single series (12.4s) were identified and
the difference between P1 and P2 (ΔP) was calculated.
is difference determines the dynamic component of
the muscle force decrease in a short period of continuous
stimulation. Mean values (mean ± SD) of the TS mus-
cle strength before and after F-injection were compared
using a two-way statistical analysis of variance (ANOVA).
e factors of variation included two conditions, time and
the effects of the C60FAS. A Bonferroni post hoc analysis
was used to determine the differences between groups.
e level of significance was set at p<0.001.
Biochemical data are expressed as the means ± SEM
for each group. e differences among experimental
groups were detected by one-way ANOVA followed by
Bonferroni’s multiple comparison test. Values of p<0.05
were considered significant.
ab
0
1
P
2
P
s
N
mm
Muscle tension (N
)
Time
5 min
Fig. 2 Strength of the contralateral (right) m. triceps surae (TS) contraction during the 2nd series of 30-min intermittent stimulations of the n. tibialis
at 42 min after C60FAS administration into the left TS (a). The superposition of individual tetanic contractions 2 and 28 are presented at the higher
time scale (b). P muscle force, st stimulation mark, L muscle length (mm); S1, S2 and S3 equal parts of the stimulus series, used for the quantitative
analysis of data; P1 and P2 sites at the beginning and at the end of tetanic contraction
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Prylutskyy et al. J Nanobiotechnol (2017) 15:8
Results
Analysis ofAFM andSANS data
Because the C60 fullerene particle size directly correlates
with their biodistribution and toxicity [29, 30], the AFM
and SANS studies were performed.
e AFM images (Fig.1a, b) clearly demonstrate ran-
domly arranged, individual C60 fullerenes (0.7 nm in
diameter) and their bulk clusters with a height of 1.5–
200nm. At the same time, some individual C60 fullerene
aggregates with a height of>200nm are also seen in the
AFM image (Fig.1b). e results obtained are consistent
with the theoretical calculations and experimental meas-
urements [20, 21, 31, 32] and demonstrate the polydis-
persity of the C60FAS used in our study.
Experimental SANS curve for C60FAS is shown in
Fig.1c. e scattering curve of C60FAS is well described
by the form-factor of polydisperse spherical particles.
e mean radius of gyration of the particle cross section,
Rg, and pair distance distribution function, P(r), were
found by using indirect Fourier transformation (IFT)
approach [33]. We can calculate the radius of particles,
R, present in the C60FAS according to well-known equa-
tion Rg
2=0.6R2 assuming of homogeneous and spherical
of C60 fullerene clusters. is conclusion follows from
previous experimental data [20, 21] and the estimates
of the average cluster density according to the contrast-
variation experiments [31, 32, 34]. e data given by this
procedure indicate that C60FAS consists of C60 fullerene
sphere-like nanoparticles with an average size of~56nm
that is in a good agreement with above AFM data.
It is known [35, 36] that the permeability and cyto-
chemical behavior of nanoparticles strongly depend on
their size and, correspondingly, mass (number) distri-
bution. In this regard, our previous studies [16, 18, 19,
29] clear demonstrate that the used C60 fullerene nano-
particles can effectively penetrate through the plasma
membrane of cells by passive diffusion or endocytosis
(depending on the size) and do not exhibit cytotoxic
effects.
Electrophysiological experiments
Changes in the TS force reaction under fatigue conditions
due to prolonged high frequency stimulation (30 min,
40/s) of the n. tibialis for animal groups 1 (before and
after administration of saline solution) and 2 (before the
application of C60FAS) did not significantly differ. e
analysis was performed by determining the force level
at the beginning (P1) and end (P2) of single tetanic con-
tractions and the difference between these values (ΔP),
which determines the dynamic component of the force
decrease during a short period of continuous stimulation
(Fig.2b). e muscle was considered tired if the ampli-
tude of the single tetanic contractions decreased by more
than 50% relative to the initial level. When muscle fatigue
was reached, the stimulation was stopped and followed
by a 10–20min rest period. erefore, in the case of one
animal, as a result of muscle fatigue stimulation of the
left TS, a 50% fatigue level was reached in approximately
12min; during the next 4min of stimulation, it contin-
ued to decrease [Fig.3a (IL), b(IL)]. After 10min of rest,
a single tetanic contraction force was slightly restored,
but it did not reach the initial muscle activity level and
continued to decrease rapidly [Fig. 3a (IIL), b(IIL)]. In
this case, there was also a simultaneous decrease in the
dynamic component of the force drop ΔP. Note that the
dynamic component was the most highly expressed at
the beginning of the first experimental series and that
the P1 amplitude was higher relative to the P2 amplitude
[Fig.3b (IIL)]. After tetanic contractions for 1.5–2min,
difference between amplitudes P1 and P2 was reduced
to zero, with moderate variations both in one and the
opposite direction over the additional fatigue stimulation
period. Simultaneously with the decrease in ΔP values,
there was a constant decrease in the developed force. In
the following stimulation series, after a period of rest, the
initial amplitude of the dynamic component was usually
decreased [Fig.3b (IIL, IIIL)].
When a predetermined level of muscle fatigue was
reached, C60FAS (0.1–0.15 mg/kg) was injected intra-
muscularly [at 45 min after the beginning of fatigue
stimulation; Fig.3a
(
I
F
L
,
IIF
L
), b(
IF
L
,
IIF
L
)]. At the same time,
the dynamic changes in the muscle strength level in
response to stimulation reflected the further develop-
ment of fatigue, and the single contraction forces were
reduced rapidly [Fig. 3b(
IF
L
)]. However, F-injection led
to the gradual recovery of the isometric force levels (at
32min after drug application; Fig.3a [(
IIF
L
), b
(
II
F
L
)]. e
appearance of negative ΔP values (P2 amplitude increase
compared to P1 amplitude) indicated the beginning of the
recovery [Fig.3b (
IIF
L
), 10th min]. In this series of stimula-
tions, the level of the muscle contraction force was recov-
ered to that developed during the initial stages of fatigue
stimulation.
Power reaction of the right TS was significantly differ-
ent from the left TS. Notably, the TS of the right limb was
not previously fatigued before the F-injection (Fig. 3c,
d). At 52 min after drug administration, a certain force
muscle decrease was observed. In this case, the P1 ampli-
tude was higher than the P2 amplitude, as indicated by
the increase in ΔP values [Fig.3c (
IF
R
), d (
IF
R
)]. However,
at 6min after the beginning of fatigue stimulation, the
force developed by the muscle appeared at a certain sta-
tionary level, which was held during the experimental
series. e difference between the P1 and P2 amplitudes
disappeared (value of ΔP decreased to zero), which may
indicate a constant force level at the time of loading. It
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Prylutskyy et al. J Nanobiotechnol (2017) 15:8
a
b
c
d
Fig. 3 Strength of the ipsi- and contralateral m. triceps surae (TS) contraction induced by electrical fatigue stimulation before and after C60FAS injec-
tion into the left TS: a, c time protocol registrations of the left and right TS contraction, respectively (triangles indicate the moment the of the C60FAS
injection); b, damplitude values of the muscle force (P1) at the beginning of single tetanic contractions (squares) and ΔP (the difference between
the force values at the beginning and at the end of muscle contraction; triangles). The rapid development of fatigue (a) (decrease in the muscle
strength of more than 50%) led to shortening of the stimulation time (I–III). Designations for I–IV (a) and I–III at (c) correspond to recordings on (b)
and (d), respectively. Indices: L, R left and right TS; F registration of muscle force after the administration of C60FAS into the left TS
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Prylutskyy et al. J Nanobiotechnol (2017) 15:8
is significant that the muscle maintained the developed
force level for an additional 1h [Fig.3c (
IIF
R
,
IIIF
R
), d(
IIF
R
,
IIIF
R
)]. For this muscle, the total time of the decrease of
the isometric force contraction by 50% was 120min after
drug administration. For comparison, the control dura-
tion of the fatigue occurrence period was 42min.
In Fig.4a, b, a comparison of the force level changes
developed by the left TS before (IL) and after (
IF
L
) F-injec-
tion in two different experiments is presented. e sta-
tistical analysis showed a significant (p < 0.001) force
decrease during the series of fatigue stimulation before
C60FAS administration [Fig.4a (IL), b(IL)]. After F-injec-
tion, a recovery of the muscle force for the first animal
[Fig.4a (
IF
L
)] and its holding for the second animal [Fig.4b
(
IF
L
)] was observed. At the end of the experimental
series, the recovery of the active muscle force response
was significant compared to that at the beginning and
nearly reached the control values. e analysis showed
a significant effect for the factors: drug administration
(D) and time (T) after administration and their interac-
tion. e corresponding results of this analysis were as
follows: F(D) = 2904.47, p(D)< 0.001, F(T)= 42.420,
p(T) < 0.001, F(DxT) = 1350.58, p(DxT) < 0.001 (first
animal) and F(D)=122.80, p(D)<0.001, F(T)=1058.29,
p(T)<0.001, p(DxT)=1287.35, p(DxT)<0.001 (second
animal). e data obtained in all experiments (Fig.4c–h)
indicate that the decrease in the developed force after
C60FAS administration (
IF
L
,
IIF
R
) was almost two times
0.00
0.50
1.00
0.00
0.50
1.00
0.00
1.00
0.50
0.00
1.00
0.50
a
c
d
e
fgh
b
ILIF
R
L
F
R
II II
Fullererne
injection
ILIR
LR
II II
Fullererne
injection
FFILIR
LR
II II
FF
ILIF
R
L
F
R
II II ILIR
LR
II II
FFILIR
LR
II II
FF
ILIF
LILIF
L
S1S2S3
0.75 0.75
0.25 0.25
**
*
***
***
Muscle tension (normalised values)
Stimulation series
Fig. 4 Averaged characteristics (mean ± SD) of normalised (to average values S1) values of the muscle strength during different parts of the fatigue
stimulation (S1, S2, and S3; Fig. 2) before and after (white and grey bars, respectively) C60FAS administration into the left m. triceps surae (TS): a, b
the results of two fatigue tests before and after C60FAS administration into the left TS; c–h the results of six fatigue tests of the left TS (open bars)
before C60FAS administration, and right TS (grey bars) at 52 min after C60FAS administration into the left TS. Asterisks significant differences (p < 0.001)
between the muscle strength during time intervals S1 and S3 in one or more series of the stimulation. I, II successive series of fatigue stimulations
(normalisation performed by S1 in series I). Indices: L, R left and right TS; F registration of muscle force after the administration of C60FAS into the left
TS. Triangle marks the moment of C60FAS injection
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Prylutskyy et al. J Nanobiotechnol (2017) 15:8
slower than in controls. e maximum significant reduc-
tion of the muscle force developed during the entire
period of fatigue stimulation was 44% after drug admin-
istration, whereas in the control this was 85%. For all
experimental animals, similar dynamics of the force level
decrease in the control and its more gradual decrease
after F-injection were observed.
Biochemical experiments
During long-term stimulation of the muscle, meta-
bolic processes change and are a main factor of muscle
fatigue. As a result of the fatigue test, the accumulation
of lipid peroxidation secondary products and changes
in the levels of antioxidants in the tissue of the fatigued
muscle were determined. e data clearly demonstrate
the increased level of peroxidation and oxidative stress
marker TBARS and H2O2 after fatigue stimulation
(Fig.5a, b). is increase was significant in relation to the
intact muscle (‘norm’) and was 23% (p<0.05) for TBARS
and 38% (p< 0.05) for H2O2. After C60FAS administra-
tion into the left TS, the TBARS concentration was sig-
nificantly reduced compared to fatigue as follows: 29%
(p<0.05) for the left TS and 12% (p<0.05) for the right
one. e H2O2 level decreases in comparison to the
‘fatigue’ group (by 6% for the left TS and 7% for the right
one), although the H2O2 level remained higher in relation
to the intact group (p<0.05). In turn, in response to such
changes in the working muscle, an activation of endog-
enous antioxidants occurred. During fatigue stimulation,
the amount of muscle GSH quantitatively increased more
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
TBARS (nM/mg protein)
norm
fatigue
C FAS (left)
60
C FAS (right)
60
0.0
0.5
1.0
1.5
2.0
НО (µМ/mg protein)
22
*
#
#
*
**
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
GSH (mМ/mg protein)
##
*
*
#
#
0.0
0.25
0.50
0.75
1.00
CAT (µМ/min/mg protein)
norm
fatigue
C FAS (left)
60
C FAS (right)
60
norm
fatigue
C FAS (left)
60
C FAS (right
)
60
norm
fatigu
e
C FAS (left)
60
C FAS (right
)
60
*
ab
cd
Fig. 5 Indicators of the prooxidant-antioxidant balance in the m. triceps surae (TS) of rats. The concentration of thiobarbituric acid reactive
substances (TBARS) (a), hydrogen peroxide (H2O2) (b), glutathione (GSH) (c) and catalase (CAT) (d) are in intact animal muscles (norm), with the
left fatigued TS (fatigue) and after C60FAS administration ipsi- and contralaterally [C60FAS (left) and C60FAS (right), correspondingly]. Values are the
mean ± SEM, n = 6. *p < 0.05 vs. “norm”; #p < 0.05 vs. “fatigue”
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Page 9 of 12
Prylutskyy et al. J Nanobiotechnol (2017) 15:8
than two-fold (p<0.05) and the activity of the antiper-
oxide enzyme CAT also increased. After C60FAS admin-
istration, the GSH and CAT activities were significantly
decreased compared to the group ‘fatigue’ by 41.8 and
15.4% for GSH and 53 and 43% for CAT (p<0.05) for the
left and right TS, respectively (Fig.5c, d).
Discussion
In this study, we investigated changes in the contraction
force of the rat m. triceps surae under fatigue develop-
ment before and after C60FAS administration. We did not
use a level of stimulation above 40Hz, and the rest period
between the experimental series was 15–20min [2]. is
experimental approach allows us to analyse the nature of
the muscle contraction force parameter changes under
fatigue stimulation before C60FAS application (into the
left TS) and directly after F-injection. A marked decrease
in the muscle effort level before C60FAS administra-
tion (control) was observe in the all experiments both IL
and IIL stimulation series (Fig.4a–h). It was the result of
modified stimulation pattern action, which was due to
the influence of the central and peripheral mechanisms
of the development of skeletal muscle fatigue [2]. After
intramuscular injection of the C60FAS partial ipsilateral
TS muscle recovery was registered in two rats. However,
the main finding was observed after the application of
C60FAS. Not significant a slower effort decrease, followed
by the prolonged retention of a certain level was recorded
contralaterally in all animals. Decrease in the muscle con-
traction force was developed more slowly after C60FAS
administration compared to the control. It indicates
a deceleration of the fatigue process, and the strength
restraint at the constant level for a long time (120min)
indicates an increase in the muscle endurance during
such conditions. e data obtained in this study indicate
that after drug injection, the time for the TS force maxi-
mal level decrease to 44% was 120min. At the same time
in the control, the force level of this muscle during the
same period decreased to 85%. We suppose, it was caused
by antioxidant effects C60FAS on the fatiguing muscle.
e duration of the muscle recovery and its rest peri-
ods are also important factors for maintaining efficiency
and the normal physiological state of the muscle during
dynamic work execution [12]. e dynamic component
of the single tetanic contraction is likely a reflection of
the interaction of the efficiency of the initial increase of
the fast motor unit contractile properties and processes
of the fatigue strength reduction [37]. us, recovery of
muscle strength after F-injection both for the preliminary
tired and at fresh muscles indicate, that water-soluble
pristine C60 fullerenes can penetrate through the plasma
membrane of cells [18, 19] and render of powerful anti-
oxidant effect on the contractile apparatus of striated
muscle, thereby facilitating its functional recovery after
experimentally induced fatigue.
Under a moderate external load on the muscle, metab-
olism occurs aerobically. In the actively contracted mus-
cle, metabolism significantly increases, resulting in the
accumulation of secondary oxidation products in muscle
fibres, which leads to fatigue development [38]. ese
metabolic processes are a source of oxygen free radicals
and contribute to the intensification of lipid peroxida-
tion processes [39–41]. e presence of such metabo-
lism products prevents the adequate implementation of
muscle work and increases the duration of the recovery
period. Strenuous exercise and endurance training cause
oxidative stress in skeletal muscle and can therefore alter
the prooxidant-antioxidant balance [42, 43]. Despite
extensive research over the years, the relationship
between free radical generation, antioxidant enzymes
and exercise in skeletal muscle remains controversial
[44, 45]. ese discrepancies may be related to differ-
ences in exercise mode, intensity, duration of the train-
ing program, and muscle fibre type. Skeletal muscles are
highly heterogeneous. Each muscle fibre type has distinct
metabolic characteristics and oxidative potential as well
as antioxidant defence capacity [41]. In our study, as a
result of fatigue stimulation in working muscle, there was
a significant increase in the secondary products of lipid
peroxidation and H2O2 compared to the intact (unstim-
ulated muscle) muscle (Fig.5). During intense (physical
activity) contraction, the flow of oxygen through muscle
cells is greatly increased. High levels of oxygen uptake
(up to 100-fold) can lead to excessive ROS generation and
are implicated in fatigue, muscle soreness, and myofibril
disruption [45]. Moreover, another potential mechanism
involved in the oxidative stress response to high-intensity
exercise is the redistribution of blood flow, such as ele-
vated blood flow in the heart, lung, and red slow-twitch
muscle fibres, leading to increased mitochondrial respi-
ration, which results in an increase in the production of
ROS. We found that long-term electrical stimulation of
the muscle induced a significant increase in TBARS and
H2O2 content that led to an increase of CAT activity and
GSH content in both fast- and slow-twitch muscle fibres.
In this case, after C60FAS administration, the oxygen
metabolite concentration was significantly lower. is
confirms the previous data regarding the protective effect
of C60FAS on the immune and antioxidant systems of the
body in various pathologies [15, 46]. e mechanisms of
effects of this drug can positively influence the processes
of endurance and recovery of the active muscles, inacti-
vating the products of its metabolism.
Increased amounts of GSH in the stimulated mus-
cle (without drug administration and after its applica-
tion) are evidence of the compensatory activation of the
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Page 10 of 12
Prylutskyy et al. J Nanobiotechnol (2017) 15:8
endogenous antioxidant systems on the irritant action
of sufficient strength (Fig.5). Many studies showed that
during intense stress, there is a significant decrease of
reduced GSH and an increased concentration of its oxi-
dative form in the myocardium and m. soleus [47, 48].
Simultaneously, contradictory data were obtained in the
experiments studying endurance [47, 49]. It was found
that under physical activity, the amount of reduced GSH
in the m. gastrocnemius and DVL increase. It is likely that
in m. soleus, a muscle with a high content of myoglobin,
all metabolic and biochemical processes occur under aer-
obic conditions, which use a large number of mitochon-
drial enzymes, and the accumulation of oxidized GSSG
does not have time to reduce [50]. At the same time,
the above mentioned processes in the m. gastrocnemius
occur anaerobically, in contrast to the m. soleus. is
causes a slow oxidation process and increases the amount
of reduced GSH [51, 52]. Under fatigue, after C60FAS
administration, the GSH content was somewhat reduced
compared to the “fatigue” state, indicating a reduction in
oxidative stress and a normalization of the pro- and anti-
oxidant balance in rat muscle tissue (Fig.5).
An increase of H2O2 during exertion leads to an
increase in CAT enzyme activity that has a protective
antioxidant function by catalysing the decomposition of
hydrogen peroxide to water and oxygen. ese results are
confirmed by previously obtained data from acute experi-
ments on rats with DVL stimulation [47, 52]. An increase
of the enzyme activity in response to exercise was also
shown in humans [53]. Moreover, some studies indicate
an absence of any changes in CAT concentration in the
muscles during physical activity [44, 54, 55]. In fact, sev-
eral reports demonstrated decreases in catalase activity
in both oxidative and mixed fibre limb muscles [56, 57].
In our study, after C60FAS administration under fatigue
development, the CAT activity was significantly reduced
compared to pure fatigue and remained at the control
level. It is hypothesized that C60FAS influence the con-
tent and activity of endogenous antioxidants and prevent
the occurrence of fatigue in actively contracting muscle,
thereby contributing to maintenance of its normal physi-
ological state.
Free radical processes increasing is the main patho-
genic factor during skeletal muscles fatigue develop-
ment [58]. Under significant physical activity there is
highly overproduction of free radicals in muscle tissue
that intensifies the processes of lipid peroxidation, cell
membranes damage and antioxidant enzymes inactiva-
tion [59]. e active oxygen metabolites cause direct
inhibition of respiratory chain mitochondrial enzymes
and reducing the balance of ATP/ADF [59]. e above
processes in the background of the lactate accumulation
with subsequent development of acidosis and blockage of
membrane Ca2+ channels lead to a pronounced energy
deficit and a significant functional activity reduction of
muscle tissue [60].
It is known that application of different nature exog-
enous antioxidants leads to a significant reduction of
fatigue skeletal muscle during intense physical activ-
ity and increases the onset time of muscle fatigue under
prolonged intense endurance exercise [10, 61, 62]. ese
data demonstrate the feasibility of using antioxidants to
correct the level of oxidative stress in the muscle tissue
under extreme influences on the body and its efficiency
increasing. Since pristine C60 fullerenes, as previously
shown in various models invitro and invivo [13, 15, 63],
actively bind free radicals and display a powerful anti-
oxidant properties of direct action, we can assume that
the application of water-soluble C60 fullerenes led to the
prooxidant-antioxidant balance normalization in the
muscle tissue of rats and helped improve the dynamic
parameters of muscle contraction.
Conclusion
e use of C60FAS, even at a low therapeutic dose (0.1–
0.15mg/kg) leads to a reduction in the recovery time of
the muscle contraction force (after its complete exhaus-
tion state) on the one hand, and an increase in the time
of the muscle active work (endurance) until fatigue devel-
opment on the other. is result illustrates the effect
of C60FAS, along with other possible mechanisms, on
prooxidant-antioxidant homeostasis in the muscle tissue
of rats.
Abbreviations
C60FAS: pristine C60 fullerene aqueous colloid solution; H2O2: hydrogen perox-
ide; TBARS: thiobarbituric acid reactive substances; GSH: reduced glutathione;
CAT: catalase; ROS: reactive oxygen species; NAD+: nicotinamide adenine
dinucleotide; HCl: hydrochloric acid; AFM: atomic force microscopy; SANS:
small-angle neutron scattering; FOX: ferrous ion oxidation xylenol orange;
H2SO4: sulphuric acid; EDTA: ethylenediaminetetraacetic acid; DAC: digital
to analogue converter; ADC: analogue to digital converter; ANOVA: analysis
of variance; DVL: deep portion of vastus lateralis muscle; GSSG: glutathione
disulfide.
Authors’ contributions
IVV, AVM and NVB designed and performed the experiments, and the in vitro
assays were performed by OOG. UR, PS and OAK were responsible for C60FAS
synthesis and characterization. TT helped with preparation of the manuscript
and provided funding support. DMN and IVM helped collect and analyze data.
YuIP and AIK provided supervision and guidance throughout this work. The
manuscript was written through contributions of all authors. All authors read
and approved the final manuscript.
Author details
1 Department of Biophysics, Taras Shevchenko National University of Kyiv,
Volodymyrska Str. 60, Kiev 01601, Ukraine. 2 Department of Movement
Physiology, Bogomoletz Institute of Physiology, Bogomoletz Str. 4, Kiev 01024,
Ukraine. 3 Department of Hypoxic States Investigation, Bogomoletz Institute
of Physiology, Bogomoletz Str. 4, Kiev 01024, Ukraine. 4 Joint Institute
for Nuclear Research, Joliot-Curie Str. 6, Dubna, Moscow Region, Russia.
5 Institute of Chemistry and Biotechnology, Technical University of Ilmenau,
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Page 11 of 12
Prylutskyy et al. J Nanobiotechnol (2017) 15:8
Weimarer Str. 25, 98693 Ilmenau, Germany. 6 University of Physical Education
and Sport, Kazimierza Górskiego Str.1, 80-336 Gdansk, Poland. 7 Lesia Ukrainka
Eastern European National University, Volya Avenue 13, Lutsk 43025, Ukraine.
Acknowledgements
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Ethics approval and consent to participate
The use of the animals was approved by the Ethics Committee of the Institute
and performed in accordance with the European Communities Council Direc-
tive of 24 November 1986 (86/609/EEC).
Funding
This work was supported by Grant 0024/RSA2/2013/52 from Rozwoj Sportu
Akademickiego, POLAND.
Received: 7 November 2016 Accepted: 30 December 2016
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