International Journal of

Molecular Sciences

Article
The Effect of Silver Nanoparticles on
Antioxidant/Pro-Oxidant Balance in a Murine Model
Anca Oana Docea 1, *,† , Daniela Calina 2, *,† , Ana Maria Buga 3,† , Ovidiu Zlatian 4,† ,
M.M.B. Paoliello 5,6 , George Dan Mogosanu 7 , Costin Teodor Streba 8 ,
Elena Leocadia Popescu 9 , Alexandra Elena Stoica 10 , Alexandra Catalina Bîrcă 10 ,
Bogdan S, tefan Vasile 10 , Alexandru Mihai Grumezescu 10 and Laurentiu Mogoanta 11
1 Department of Toxicology, University of Medicine and Pharmacy of Craiova, 200349 Craiova, Romania
2 Department of Clinical Pharmacy, University of Medicine and Pharmacy of Craiova, 200349 Craiova,
Romania
3 Department of Biochemistry, University of Medicine and Pharmacy of Craiova, 200349 Craiova, Romania;
[email protected]
4 Department of Microbiology, University of Medicine and Pharmacy of Craiova, 200349 Craiova, Romania;
[email protected]
5 Graduate Program in Public Health, Center of Health Sciences, State University of Londrina, 60 Robert Koch
Avenue, Londrina 86038-350, Brazil; [email protected]
6 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Forchheimer 209,1300 Morris
Park Avenue, Bronx, NY 10461, USA
7 Department of Pharmacognosy and Phytotherapy, Faculty of Pharmacy University of Medicine and
Pharmacy of Craiova, 200349 Craiova, Romania; [email protected]
8 Department of Research Methodology, University of Medicine and Pharmacy of Craiova, 200349 Craiova,
Romania; [email protected]
9 Doctoral School University of Medicine and Pharmacy of Craiova, 200349 Craiova, Romania;
[email protected]
10 Department of Science and Engineering of Oxide Materials and Nanomaterials, Faculty of Applied
Chemistry and Materials Science, Politehnica University of Bucharest, 011061 Bucharest, Romania;
[email protected] (A.E.S.); [email protected] (A.C.B.); [email protected] (B.S.V.);
[email protected] (A.M.G.)
11 Department of Histology, University of Medicine and Pharmacy of Craiova, 200349 Craiova, Romania;
[email protected]
* Correspondence: [email protected] (A.O.D.); [email protected] (D.C.)
† These authors contributed equally to this work.




Received: 31 December 2019; Accepted: 7 February 2020; Published: 12 February 2020


Abstract: This study aimed to evaluate the subacute effect of two types of Ag-NPs(EG-AgNPs
and PVP-EG-AgNPs) on antioxidant/pro-oxidant balance in rats. Seventy Wistar rats (35 males
and 35 females) were divided in 7 groups and intraperitoneally exposed for 28 days to 0, 1, 2
and 4 mg/kg bw/day EG-Ag-NPs and 1, 2 and 4 mg/kg bw/day PVP- EG-Ag-NPs. After 28 days,
the blood was collected, and the total antioxidant capacity (TAC), thiobarbituric reactive species
(TBARS),protein carbonyl (PROTC) levels, reduced glutathione (GSH) levels and catalase (CAT)
activity were determined. EG-Ag-NPs determined protective antioxidant effects in a dose-dependent
manner. The exposure to the 4 mg/kg bw/day EG-Ag-NPs determines both in males and females
a significant increase in TAC and CAT and a significant decrease in TBARS and PROTC only in
females. The PVP-EG-AgNPs showed a different trend compared to EG-AgNPs. At 4 mg/kg
bw/day the PVP-EG-AgNPs induce increased PROTC levels and decreased GSH (males and
females) and TAC levels (males). The different mechanisms of EG-AgNPs and PVP-EG-AgNPs on
antioxidant-/pro-oxidant balance can be explained by the influence of coating agent used for the
preparation of the nanoparticles in the formation and composition of protein corona that influence
their pathophysiology in the organism.

Int. J. Mol. Sci. 2020, 21, 1233; doi:10.3390/ijms21041233 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 1233 2 of 17

Keywords: silver nanoparticles; oxidative stress; antioxidant activity; subacute toxicity

1. Introduction
Nanoparticles (NPs) have multiple applications in biomedicine, such as controlled drug
administration and diagnostic applications [1–7]. Silver nanoparticles (AgNPs) showed many
beneficial effects mainly due to their antibacterial effects, both gram-negative and gram-positive,
being effective also against strains with a high degree of virulence [8–12]. With the development of
nanotechnology and the use of nanoparticles especially in the medical and pharmaceutical fields, the
need for investigation of their toxic effects is essential. Nanoparticles used in the pharmaceutical field
have several advantages in improving the targeted delivery of drugs and decreasing their toxicity.
Furthermore, metal nanoparticles can be combined with infrared light, radio waves or magnetic field
and used for thermal ablation of diseased tissues [13–15].
Taking into consideration all the advantages of nanoparticles, evaluation of their toxicity is critical.
In order to approve a nano-formulation, a complete pharmacological and toxicological profile is
essential [16–18].
Recently, several silver nano-formulations have been designed and tested for their potential
pharmacological effects as potent antibacterial drugs [7,19–21] and promising antitumor agents [22,23].
These nano-formulations differ in size, shape, and surface coating [24–26].
Until now, several studies investigated the antibacterial mechanism of action of Ag-NPs. This
mechanism is influenced by several factors as NPs diameter, shape, surface changes. Sharma etal. have
shown that the antibacterial effect of Ag-NPs is produced in a dose-dependent manner, mainly against
gram-negative bacteria, and this antibacterial effect is independent of the acquirement germ resistance
to antibiotics. The main mechanisms by which the silver nanoparticles showed their antibacterial
properties were by fixation and penetration of the cell wall and modulation of cell signaling [27].
Pal et al. demonstrated that silver nanoparticles interact differently, depending on the shape, with
Escherichia coli. Thus, truncated triangular silver nanoparticles had the highest biocidal activity,
compared to spherical and rod nanoparticles and ionic silver [28]. Morones et al. [29] used different
types of gram-negative bacteria to test the antibacterial activities of silver nanoparticles in the 1–100 nm
range. Antibacterial activity of Ag-NPs against gram-negative bacteria has been shown to be divided
into three steps: (1) nanoparticles with dimensions of 1–10 nm are capable todrastically disrupt normal
functions of bacteria, such as permeability and respiration by attaching to the surface of the cell
membrane; (2) these nanoparticles are capable of penetrating into the bacterium and causing further
damage through possible interaction with compounds containing sulfur and phosphorus, such as
DNA; (3) the nanoparticles release silver ions, which will further contribute to the bactericidal effect
of the silver nanoparticles [29]. Smekalova et al. reported that the antibacterial activity of silver
nanoparticles also depends on surface changes (surfactants/polymers) [30]. As a summarization, the
three best-known antibacterial mechanisms of AgNPs are (1) silver ion uptakeby the bacterial cell,
followed by disruption of ATP production and DNA replication, (2) generation of reactive oxygen
species (ROS) by silvernanoparticles and silver ionsand (3) direct damage of cell membranes by silver
nanoparticles. However, further investigations are needed to clarify these mechanisms, especially the
issue of the affinity of silver nanoparticles for bacterial proteins containing sulfur and phosphorus and
the effects of this affinity on bacterial protein functions [31].
Regarding the use of Ag-NPs as promising antitumor agents, studies have been shown that these
effects are correlated with the induction of oxidative and nitro-oxidative stress in cancer cells that lead
to mitochondrial disruption and cancer cell death [32].
Int. J. Mol. Sci. 2020, 21, 1233 3 of 17

Their physical and chemical properties also influence their toxicological profile [25]. Route of
administration, dose and exposure are critical factors that affectthe degree of toxicity produced by a
particular type of NP. In the case of soluble nanoparticles, their toxicity is governed by the components,
while in the case of insoluble nanoparticles as stable metal oxides, the mechanism is more complex.
Silver nanoparticles (Ag-NPs) have been shown to act by inducing oxidative stress that leads to
cytotoxic and genotoxic effects through the induction of DNA damage and apoptosis [33–35].These
effects depend on colloidal stability and the cellular uptake of the NPs that is influenced by the coated
agent, NPs diameter and by the doses used, usually above 10 mg/kg bw in rodents.
In the synthesis of Ag-NPs, the coating process helps in enhancing the stability in the solution
by decreasing their agglomeration and preventing the cytotoxicity of Ag-NPs against the living cells,
being important factors in decreasing the toxicity of Ag-NPs [36]. Several coating methods have
been used for the preparation of Ag-NPS as polymerization, sol-gel method, successive ionic layer
absorption and reaction (SILAR) method or biomolecule-mediated Ag-NPs organization. As coating
agents in the literature, we find two big categories: organic substances and inorganic substances as
metals, metal oxides and metal salts [37]. Ag-NPs have been synthesized using as organic capping
agents citric acid, polymers, proteins, polysaccharides, surfactants, etc. From polymers polyethylene
glycol and polyvinylpyrrolidone (PVP) are mainly used for stabilizing the AgNPs [38]. The coated
Ag-NPs act differently on the organism compared to the uncoated Ag-NPs;hence, their toxicity hasto
be evaluated independently in vitro and in vivo.
The generation of ROS by Ag-NPs had a double impact on the therapeutic utilization of these
NPs as this mechanism is implicated both in therapeutical efficacy and toxicity. In this study, we
synthesized two types of Ag-NPs, one functionalized with ethylene glycol (EG-Ag NPs) and the other
functionalized with polyvinylpyrolidone and ethylene glycol (PVP-EG-Ag NPs), and we evaluated the
subacute (28 days of intraperitoneal administration) effect on antioxidant/pro-oxidant balance in rats
as NPs effects on this can influence both the toxicity and the clinical efficacy of Ag-NPs.

2. Results

2.1. Transmission Electron Microscopy (TEM) and Selected Area (Electron) Diffraction (SAED)
EG-AgNPs and PVP-EG-AgNPs samples were characterized by Transmission Electron Microscopy;
the images obtained are presented in Figure 1. Analyzing the TEM images presented for the two
experimental variants, particles of nanometric dimensions can be observed, crystalline, covered by
a phase that has low crystallinity that corresponds to the organic components used in the obtaining
process (EG and PVP). The EG-AgNPs have an average diameter of 9.44 nm with a Zeta Potential of
−14.49 mV, while PVP-EG-AgNPs particles have an average diameter of 16.89 nm (Figure 2) with a
Zeta Potential of −47.94 mV, and the coating has thicknesses of 1–3 nm.
Int. J. Mol. Sci. 2020, 21, 1233 4 of 17
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 16

Figure 1.images recorded for (a) EG-AgNPs and (b) PVP-EG-AgNPs.

Figure 1. images recorded for (a) EG-AgNPs and (b) PVP-EG-AgNPs.
Int. J. Mol. Sci. 2020, 21, 1233 5 of 17
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 16

Figure 2.
Figure 2.Histogram
Histogramindicating
indicating the
the particle
particle size
size distribution:
distribution: (a)Ag-NPs
(a)Ag-NPs functionalized
functionalized with
with ethylene
ethylene
glycol (EG-Ag
glycol (EG-Ag NPs);
NPs); (b)Ag-NPs
(b)Ag-NPs functionalized
functionalized with
with polyvinylpyrolidone
polyvinylpyrolidone and
and ethylene
ethylene glycol.
glycol.

2.2. The
TheEffect
SAED of EG-AgNPs on Anti-Oxidant
pattern provides Markers
information about the crystallinity of the characterized samples
(EG-AgNPs and PVP-EG-AgNPs). In the case of both experimental variants, it is observed, from the
2.2.1. TAC
analysis Levels
of the diffraction rings (identified and measured), that the only crystalline phase present is the
hexagonal silver. It has beenafter
TAC levels increased identified andto
exposure corresponds
EG-AgNPstointhe
allICDD file [PDF
the treated card no.
groups 01-071-5025].
compared to the
control group both in males and females. The increase was not dose-dependent. The females treated
2.2. The Effect of EG-AgNPs on Anti-Oxidant Markers
with 2 mg/kg bw showed a higher increase expressed as a percentage compared to the control
(135.3%),
2.2.1. TACfollowed
Levels by female rats treated with 1 mg/kg bw (122.8%) and the 4 mg/kg bw (11.6%), as
shown in Table 1 (p <0.05). The same trend was also observedin male rats, in 1 mg/kg bw group, the
TAC levels increased after exposure to EG-AgNPs in all the treated groups compared to the control
TAC levels increased compared to the control group with 106.2%(p <0.05), while in 2 mg/kg bw and
group both in males and females. The increase was not dose-dependent. The females treated with
4 mg/kg bw groups the increase was with 55.2%(p <0.05)and with 5.7%(p >0.05), respectively (Table
2 mg/kg bw showed a higher increase expressed as a percentage compared to the control (135.3%),
1).
followed by female rats treated with 1 mg/kg bw (122.8%) and the 4 mg/kg bw (11.6%), as shown in
Table 1 (p < 0.05).
Table The same trend
1.Markers wasafter
at 28 days alsoexposure
observedin male rats,
to different in 1 mg/kg of
concentrations bwEG-AgNPs.
group, the TAC levels
increased compared to the control group with 106.2%(p < 0.05), while in 2 mg/kg bw and 4 mg/kg bw
Males Females
groups the increase was with 55.2%(p < 0.05)and with 5.7%(p > 0.05), respectively (Table 1).
Parameter 1 2 4 1 2 4
Control Control
mg/kgbw mg/kgbw mg/kgbw mg/kgbw mg/kgbw mg/kgbw
Table 1. Markers
Average 0.2422 at 28 days* after0.3758
0.4993 exposure
* to0.2561
different concentrations
0.2316 0.5161 of*EG-AgNPs.
0.5450 * 0.2584 *
TAC
SD 0.0138 0.492 0.0492 0.0157 0.0159 0.0091 0.0140 0.0060
(mmol Males Females
% to
DPPH/L)
Parameter 106.2% 1 55.2% 2 5.7% 4 122.8% 1 135.3%
2 11.6%
4
control Control Control
mg/kgbw mg/kgbw mg/kgbw mg/kgbw mg/kgbw mg/kgbw
Average 1.9408 1.8092 1.9901 2.0559 2.2039 2.4178* 1.9342* 2.2270
GSH
TAC SD Average0.1628 0.24220.06010.4993 *0.03490.3758 * 0.03490.2561 0.0233
0.2316 0.5161 *
0.0814 0.5450
0.0221* 0.2584
0.1506*
(µmol/g SD 0.0138 0.492 0.0492 0.0157 0.0159 0.0091 0.0140 0.0060
(mmol DPPH/L) % to% to control
Hb) −6.8% 106.2% 2.5%55.2% 5.9%5.7% 122.8%
9.7% 135.3%
−12.2% 11.6%
1.1%
controlAverage 1.9408 1.8092 1.9901 2.0559 2.2039 2.4178* 1.9342* 2.2270
GSH
Average
(µmol/g Hb)
SD177.38 0.1628
168.5714 0.0601 185.020.0349 192.96 0.0349 171.03
0.0233 171.10
0.0814 177.78
0.0221 188.27*
0.1506
CAT SD % to control
2.38 28.9076−6.8% 2.175 2.5% 1.615.9% 0.56 9.7%
2.51 −12.2%
1.56 1.1%
11.25
(U/g Hb)CAT % to Average 177.38 168.5714 185.02 192.96 171.03 171.10 177.78 188.27*
SD 2.38−5.0% 28.9076 4.3% 2.175 8.8%1.61 0.56 0.1%
2.51 3.9%
1.56 10.1%
11.25
(U/g Hb) control
% to control −5.0% 4.3% 8.8% 0.1% 3.9% 10.1%
Notes:
Notes:* p<
* p<0.05
0.05compared tothe
compared to thecontrol
control group.
group.

2.2.2. GSH Levels

2.2.2.Regarding
GSH LevelsGSH levels, the trend was slightly different in males compared with females. In
males,a decreaseGSH
Regarding of GSH levels
levels, thewas
trendobserved in 1 mg/kg
was slightly bw group
different compared
in males comparedto control and thenIna
with females.
dose-dependent increase in 2 and 4 mg/kg bw groups without reaching statistical
males, a decrease of GSH levels was observed in 1 mg/kg bw group compared to control and thensignificance. In
females, an increase of GSH levels was observed first compared to control in 1 mg/kg
a dose-dependent increase in 2 and 4 mg/kg bw groups without reaching statistical significance. Inbw group,
without reaching
females, theofstatistical
an increase GSH levelssignificance, then first
was observed a decrease compared
compared to control
to control in 2 mg/kg
in 1 mg/kg bw
bw group,
group (p <0.05) followed by a slight increase compared to control in 4 mg/kg bw group (p >0.05) (Table
1).
Int. J. Mol. Sci. 2020, 21, 1233 6 of 17

without reaching the statistical significance, then a decrease compared to control in 2 mg/kg bw group
(p < 0.05) followed by a slight increase compared to control in 4 mg/kg bw group (p > 0.05) (Table 1).

2.2.3. CAT Activity Levels

CAT levels decreased in the 1 mg/kg bw group treated males compared to control and then
increased in a dose depending manner in 2 and 4 mg/kg bw group, but without reaching the statistical
significance. In females, we observed a dose-dependent increase of CAT levels compared to control in
1, 2 and 4 mg/kg bw group, respectively, reaching the statistical significance only in 4 mg/kg bw group
(Table 1).

2.3. The Effect of PVP-EG-AgNPs on Anti-Oxidant Markers

2.3.1. TAC Levels

Exposure to PVP-EG-AgNPs showed different effects on TAC levels in male compared to female
rats. In male rats exposed to 1 mg/kg bw PVP-EG-AgNPs, increased TAC levels were noted compared
to the control (43.4%)(p < 0.05), while treatment with 2 mg/kg bw or 4 mg/kg bw PVP-EG-AgNPs led
to decreased TAC levels compared with the controls (5.1 and 15.9%, respectively), reaching statistical
significance only for 4 mg/kg bw group(Table 2). In female rats the nanoparticle treatment led to 73.1%
increase in TAC levels compared to controls after exposure to 1 mg/kg bw PVP-EG-AgNPs (p < 0.05),
followed by a decrease after exposure to 2 mg/kg bw or 4 mg/kg bw PVP-EG-AgNPs compared to the
control, but without reaching the statistical significance (p > 0.05) (Table 2).

Table 2. Markers at 28 days after exposure to different concentrations of PVP-EG-AgNPs.

Males Females
Parameter 1 2 4 1 2 4
Control Control
mg/kgbw mg/kgbw mg/kgbw mg/kgbw mg/kgbw mg/kgbw
Average 0.2422 0.3472 * 0.2545 0.2038 * 0.2316 0.4009 * 0.2171 0.2450
TAC
SD 0.0138 0.0016 0.0138 0.0063 0.0159 0.0315 0.0031 0.0039
(mmol DPPH/L)
% to control 43.4% −5.1% −15.9% 73.1% −6.3% 5.8%
Average 1.9408 2.2862 2.1875 0.8224 * 2.2039 2.1382 2.0066 * 1.5461 *
GSH
SD 0.1628 0.1977 0.1047 0.3489 0.0233 0.1163 0.0930 0.0698
(µmol/g Hb)
% to control 17.8% 12.7% −57.6% −3.0% −9.0% −29.9%
Average 177.38 169.84 216.47* 181.84 171.03 199.01* 188.09 179.66
CAT
SD 2.38 5.61 15.43 2.59 0.56 14.73 5.33 13.96
(U/g Hb)
% to control −4.3% 22.0% 2.5% 16.4% 10.0% 5.1%
Notes: * p< 0.05 compared to control group.

2.3.2. GSH Levels

The nanoparticle treatment influences the GSH levels in a different manner between males and
females. In males, the exposure to 1 and 2 mg/kg bw PVP-EG-AgNPs determines an increased
compared to controlwithout reaching the statistical significance (p > 0.05), while theexposure to 4 mg/kg
bw PVP-EG-AgNPs decreased the GSH levels compared to control(p < 0.05) (Table 2). In females, the
nanoparticle treatment determined a dose-dependent decrease in GSH levels compared to control after
treatment with 1, 2 and 4 mg/kg bwPVP-EG-AgNPs respectively, reaching the statistical significance
only for 2 and 4 mg/kg bwgroups (p < 0.05)(Table 2).

2.3.3. CAT Activity Levels

The effects of PVP-EG-AgNPs administration on CAT levels determine in males from 1 mg/kg bw
group a decrease compared to control followed by an increase compared to control in 2 and 4 mg/kg
bw groupsPVP-EG-AgNPsreaching the statistical significance only for 2 mg/kg bw group (Table 2).
In females, the treatment with PVP-EG-AgNPs led to an increased compared to the control in all the
treated groups, reaching the statistical significance only for 1 mg/kg bwgroup (p < 0.05) (Table 2).
Int. J. Mol. Sci. 2020, 21, 1233 7 of 17

2.4. The Effect of EG-AgNPs on Pro-Oxidant Markers

2.4.1. TBARS Levels

The TBARS levels increased both in malesand females after treatment with 1 mg/kg bw EG AgNPs
compared to control with 77.65% and 1.22%, respectively, reaching the statistical significance only for
males (Table 3). Exposure to 2 mg/kg bw EG AgNPs and 4 mg/kg bw EG AgNPs led to decreased
TBARS levels compared to controls both in males and in females, but the statistical significance is
reached only for females (Table 3).

Table 3. Markers at 28 days after exposure to different concentrations of EG-AgNPs.

Males Females
Parameter 1 2 4 1 2 4
Control Control
mg/kgbw mg/kgbw mg/kgbw mg/kgbw mg/kgbw mg/kgbw
Average 0.6290 1.1174 * 0.5323 0.4903 0.7032 0.7118 0.4361 * 0.4619 *
TBARS
SD 0.0798 0.3934 0.0205 0.0274 0.0274 0.0249 0.0202 0.0195
(µmol/L)
% to control 77.7% −15.4% −22.1% 1.2% −38.0% −34.3%
PROTC Average 0.9008 1.0297 0.9450 0.8287 1.0792 1.7134* 0.7076 0.5934*
(nmol/mg SD 0.1029 0.0649 0.1090 0.1711 0.0611 0.7115 0.0257 0.1499
protein) % to control 14.3% 4.9% −8.0% 58.8% −34.4% −45.0%
Notes: * p< 0.05 compared to thecontrol group.

2.4.2. PROTC Levels

Treatment with EG-AgNPs determined in male rats increased PROTC levels compared to controls
in 1 mg/kg bw group and 2 mg/kg bw group and then a decrease compared to control in 4 mg/kg bw
groupwithout reaching the statistical significance (Table 3). In female rats, it was observed an increase
in PROTC levels compared to control in 1 mg/kg bw group (p < 0.05) and then a decrease compared to
control in 2 mg/kg bw and 4 mg/kg bw groups, reaching the statistical significance only for 4 mg/kg bw
group (Table 3).

2.5. The Effects of PVP-EG-AgNPs on Pro-Oxidant Markers

2.5.1. TBARS Levels

The exposure to PVP-EG-AgNPs led to a dose-dependent decrease in TBARS levels compared to
control group in males, reaching statistical significance only in 2 and 4 mg/kg bw groups, while in
females the highest decrease was reached by the 1 mg/kg bw groups, followed by 2 mg/kg bwthat
reached the statistical significanceand 4 mg/kg bw group (p < 0.05), respectively (Table 4).

Table 4. Markers at 28 days after exposure to different concentrations of PVP-EG-AgNPs.

Males Females
Parameter 1 2 4 1 2 4
Control Control
mg/kgbw mg/kgbw mg/kgbw mg/kgbw mg/kgbw mg/kgbw
Average 0.6290 0.6710 0.4968* 0.5194* 0.7032 0.4677* 0.5065* 0.8129
TBARS
SD 0.0798 0.0502 0.0137 0.0068 0.0274 0.0205 0.0205 0.1962
(µmol/L)
% to control −6.7% −21.0% −17.4% −33.5% −28.0% 15.6%
PROTC Average 0.9008 0.6718 2.1341* 3.8725* 1.0792 0.7884 2.5592* 2.5274*
(nmol/mg SD 0.1029 0.0516 0.8640 0.1741 0.0611 0.0320 0.0994 0.4932
protein) % to control −25.4% −136.9% 330.0% −27.0% 137.1% 134.2%
Notes: * p< 0.05 compared to the control group.

2.5.2. PROTC Levels

In males, treatment with PVP-EG-AgNPs led to decreased PROTC levels compared to controls at 1
(p > 0.05) and 2 mg/kg bw (p < 0.05), respectively, and then an increase compared to control in 4 mg/kg
Int. J. Mol. Sci. 2020, 21, 1233 8 of 17

bw group (p < 0.05) (Table 4). In females was observed a decrease compared to control after exposure
to 1 mg/kg bw PVP-EG-AgNPs, without reaching a statistical significanceand then an increase in the 2
and 4 mg/kg bw PVP-EG-AgNPs (p < 0.05), respectively (Table 4).

3. Discussion
The extended use of AgNPs in the pharmaceutical field has raised concernsregarding their health
effects. Several studies have investigated the mechanism of toxicity of Ag –NPs suggesting that their
cytotoxicity is mainly mediated by induction of reactive oxygen species (ROS). The size of the NPs,
their dose and the duration of treatment are critical in mediating their effects [39,40]. Another factor
associated with the cytotoxicity of AgNPs is the surface-stabilizing agent [41,42].
In this study, we used two different coatings for the synthesis of nanoparticles: EG and PVP-EG.
The aim of this study was to investigate the effect of subacute administration of EG-Ag-NPs and
PVP-EG-Ag-NPs (for 28 days) on antioxidant/pro-oxidant balance in rats. The antioxidant/pro-oxidant
balance was evaluated by determining the levels of TAC, TBARS, PROTC, GSH and CAT. TAC is the
marker that evaluates the antioxidant status of the biological system and is very useful to evaluate the
response of the organism against the free radical production [43]. TBARS is one of the oldest and most
widely used markers for the evaluation of lipid peroxidation [44]. PROTC is a marker that evaluates
the oxidation of the proteins that increase due to oxidative stress [45]. Reduced GSH is considered to
be the first line of non-enzymatic antioxidant of the defense system that fights against oxidative stress
and gets depleted in the oxidative stress models [46]. CAT is the enzyme that converts superoxide
to water and molecular oxygen and prevents the formation of hydroxyl radical and other toxic ROS
species [47].
We showed that the exposure of rats to EG-Ag-NPs determined protective antioxidant effectswith
slight differences between males and females. At doses of 1 mg/kg bw/day, a significant increase in
TAC levels associated with a significant increase in TBARS in maleswas observed.In females exposed to
1 mg/kg bw/day, a significant increase in TAC, GSH and PROTC levelswas observed. An explanation
for this discrepancy between TBARS level in males and females at the same dose can be explained by
the gender-dependent response of the body that affects in a specific way the oxidative damage of the
lipids. It is well known that females seem to respond faster than males to oxidative stress displaying
increased protection due to estrogen [48–50]. However, further investigation should be performed in
order to elucidate the mechanism that is involved in a gender-dependent response to lipid peroxidation.
At the medium, dose tested, the protective effects of the EG-Ag-NPs are more visible especially in
females where we observed a significant increase in TAC levels and a significant decrease of TBARS.
The exposure to the 4 mg/kg bw/day EG-Ag-NPs determines protective effects against oxidative stress,
especially in females, translated by a significant increase of TAC and CAT and a significant decrease in
TBARS and PROTC in females. The same trend was present also in males but without reaching the
statistical significance. The difference in the patterns observed in males and females can be explained by
the gender differences in circulation and elimination of AgNPs [51]. The protective antioxidant effects
observed for EG-AgNPs are in line with the study of Singh et al. (2018) that showed the protective
effect of AgNPS against chemical-induced hepatotoxicity in rats by re-establishing the antioxidant
levels [7]. Antioxidant effects of AgNPsareone of their beneficial effects, which determined their use in
a lot of products for biomedical application [52]. AgNPs synthesized by green nanotechnology showed
to have antioxidant activity [53,54]. PatilShriniwas et al. (2017) showed that AgNPs synthesized using
terpenes extracted from Lantana camara L. showed, at doses of 2 mg/mL, antioxidant activity similar
to ascorbic acid [55]. It is also worth investigating if these protective effects against oxidative stress
are valid in the real-life exposure scenario where the individual is exposed in a chronic manner to a
combination of stimuli that can be additive, synergic or antagonist [56,57]. Several studies showed that
the combined exposure at low doses can produce non-monotonic responses [58–60], and this is worth
investigating also for Ag-NPs as the exposure to these molecules usually appearsin combination to
other agents that can influence their effects on biological organisms.
Int. J. Mol. Sci. 2020, 21, 1233 9 of 17

The PVP-EG-AgNPs showed a different trend regarding antioxidant/pro-oxidant balance compared

to EG-AgNPs.At higher doses, the NPs induce increased protein oxidation translated by increased
PROTC levels and decreased GSH both in males and females and TAC levels in males after exposure
to 4 mg/kg bw/day. Patlolla et al. showed that evaluating the acute toxicity of oral administration of
AgNPs at doses of 50 and 100 mg/kg bw/day, the ROS induction increased compared to the control
group [61]. Foldbjerg et al. showed that PVP-coated AgNPs dramatically increased ROS levels in
the human monocytic cell line that led to cell apoptosis and necrosis [62]. The mechanism by which
the Ag-NPs induce oxidative stress seems to be associated with the generation of intracellular ROS
determined by the Ag+ ions found on the surface of the NPs or released from the NPs [23,62,63]. This
is also supported by our findings that showed increased protein oxidation translated by increased
PROTC levels. Another possible mechanism of Ag-NPs induced oxidative stress is mediated by the
affinity of the NPs to the thiol groups, which lead to a reduction of GSH levels and the inability of this
molecule to neutralize the ROS [64]. This effect is also supported by our findings, which showed that
exposure to PVP-EG-AgNPs produces a decrease in GSH and TAC levels. These results can contribute
to the further investigation of the beneficial effects of synthesized PVP-EG-AgNPs in cancer treatment.
Ag-NPs have been shown to induce programmed cell death in several cancer cell lines, and these
effects have been correlated with the induction of ROS species [23,32].
The coating of Ag-NPs is used to stabilize the NPs by producing electrostatic and electrosteric
repulsions between particles. Several studies showed that coating also protects against the cytotoxicity
manifested by Ag-NPs [65]. It was demonstrated that the coating of nanoparticles influences the
formation and composition of protein corona in the biological fluid that can further influencethe
pathophysiology of the NPs in vitro [18,66]. In our study, we demonstrated that the coating agent
affects the effects of Ag-NPs on antioxidant/pro-oxidant balance and can be an essential factor in
Ag-NPs toxicityor targeted therapeutic effect of Ag-NPs.

4. Materials and Methods

4.1. Raw Materials

Silver nitrate (AgNO3), Sodium hydroxide (NaOH), Ethylene glycol (EG) and Polyvinylp
yrrolidone (PVP) were purchased from Sigma–Aldrich without further purification. All chemicals
were of analytical purity and used with no further purification. Deionized water was used throughout
the experiment.

4.2. Synthesis of EG-AgNPs and PVP-EG-AgNPs

The experimental obtentionof silver nanoparticles in the presence of EG and EG/PVP was possible
by using a chemical method to reduce the metal precursor, which involved obtaining two solutions
necessary for the synthesis process for each particular synthesis. The silver nitrate solutions were
obtained by dissolving 1 g of AgNO3 in 300 mL of ultrapure water. The second solution was
prepared by dissolving 20 g NaOH and 3 g EG and 1,5 g EG + 1,5 g PVP, respectively, in 400 mL
ultrapure water under magnetic stirred at 80◦ C. Subsequently, silver nitrate solutions were added
to the polymer/monomer solutions by dripping and under continuous magnetic stirring. During
the process, it was observed the change in the color of the dispersions obtained an aspect correlated
with the formation of silver particles. The collection of the synthesized nanoparticles was possible
by vacuum filtration of the obtained dispersions. The silver particles were subjected to a triple wash
treatment with distilled water and dried at room temperature (see Figure 3).
Int. J. Mol. Sci. 2020, 21, 1233 10 of 17
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 16

Figure 3.Representation
Figure 3. Representationof
ofEG-Ag-NPs
EG-Ag-NPssynthesis.
synthesis.

4.3. Characterization
4.3. Characterization of
of EG-AgNPs
EG-AgNPs and
and PVP-EG-AgNPs
PVP-EG-AgNPs

4.3.1. Transmission
4.3.1. Transmission Electron
Electron Microscopy
Microscopy (TEM)
(TEM)
TEM images
images were
were obtained TM G2 F30 S-TWIN transmission
TEM obtained using
using aa high-resolution
high-resolution Tecnai
TecnaiTM G2 F30 S-TWIN transmission
microscope equipped
microscope equipped with
with SAED,
SAED, from
from the
the FEI
FEI (Oregon,
(Oregon, USA).
USA). The
The transmission
transmission modewas
modewas used
used at
at
300 kV,
300 kV, the
the point
point and
and line
line resolutions
resolutions were
were 22 Å
Å and
and 11 Å,
Å, respectively.
respectively.
4.3.2. Zeta Potential Measurement
4.3.2. Zeta Potential Measurement
Zeta potential measurements were performed using a DelsaMax Pro equipped with a laser at
Zeta potential measurements were performed using a DelsaMax Pro equipped with a laser at
532 nm. The samples were prepared by dispersing in ultrapure water at room temperature.
532 nm. The samples were prepared by dispersing in ultrapure water at room temperature.
4.4. Animals
4.4. Animals
Seventy Wistar rats (35 males and 35 females), 12 weeks old, with a median weight of 343 ± 20 g
Seventy Wistar rats (35 males and 35 females), 12 weeks old, with a median weight of 343± 20 g
for males and 236 ± 22 g for females were obtained from the Animal House of the University of
for males and 236 ± 22 g for females were obtained from the Animal House of the University of
Medicine and Pharmacy of Craiova, Craiova, Romania. One week before the start of the study, the
Medicine and Pharmacy of Craiova, Craiova, Romania. One week before the start of the study, the
animals were acclimatized to the new conditions with constant temperature 22 ± 2◦ C, humidity
animals were acclimatized to the new conditions with constant temperature 22 ± 2°C, humidity
between 40% to 60% and dark/light cycle of 12 h/12 h. The animals received food and water ad libitum.
between 40% to 60% and dark/light cycle of 12 h/12 h. The animals received food and water ad
The animal experiment was approved by the Ethical Committee of the University of Medicine and
libitum. The animal experiment was approved by the Ethical Committee of the University of
Pharmacy of Craiova, Craiova, Romanianumber 89/13.09.2018 and respected all the directives for
Medicine and Pharmacy of Craiova, Craiova, Romanianumber 89/13.09.2018 and respected all the
animal experiments requested by EU Commission Directive 2010/63/EU.
directives for animal experiments requested by EU Commission Directive 2010/63/EU.

4.5. Experimental Design

The rats were assigned to 7 groups (5 males and 5 females per group), one control and 6
treatment groups as depicted in Figure 4. The treatment groups were treated with the dispersion of
nanoparticles in ultrapure water at room temperature.
Int. J. Mol. Sci. 2020, 21, 1233 11 of 17

4.5. Experimental Design

The rats were assigned to 7 groups (5 males and 5 females per group), one control and 6 treatment
groups as depicted in Figure 4. The treatment groups were treated with the dispersion of nanoparticles
in ultrapure water at room temperature.
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 16

Figure
Figure4.Design of the
4. Design study.
of the study.

After 28
After 28days
daysofofexposure,
exposure,thethe rats
rats were
were immobilized
immobilized with
with a restrainer
a restrainer and and the blood
the blood was
was collected
collected
into into ethylenediaminetetraacetic
ethylenediaminetetraacetic acid (EDTA)acid (EDTA) vacutainers
vacutainers from
from tail tail veins
veins usingusing a 23
a 23 G G needle.
needle.

4.6. Oxidative
4.6. Oxidative Stress
Stress Markers
MarkersEvaluation
Evaluation
All the
All the reagents
reagents for for oxidative
oxidativemarkers
markerswere werepurchased
purchased from
fromSigma-Aldrich
Sigma-Aldrich (USA). The The
(USA). blood blood
samplescollected
samples collected inin EDTA
EDTA werewere centrifuged
centrifugedatat1370×g
1370×for g for10 10
min at 4at°C,
min 4 the
◦ plasma
C, the and and
plasma erythrocytes
erythrocytes
were separated
were separated and and stored
stored at at −80
−80°C ◦ Cfor
forfurther
furtheranalysis.
analysis. TheThetotal antioxidant
total antioxidant capacity (TAC),
capacity (TAC),
thiobarbituric reactive species (TBARS) and protein carbonyl (PROTC)
thiobarbituric reactive species (TBARS) and protein carbonyl (PROTC) levels were determined levels were determined in in
plasma as previously described [58,67,68]. The collected packed erythrocytes were lysed with
plasma as previously described [58,67,68]. The collected packed erythrocytes were lysed with distilled
distilled water (1:1 v/v) followed by centrifugation at 4020×g at 4 °C for 15 min and then the
water (1:1 v/v) followed by centrifugation at 4020× g at 4 ◦ C for 15 min and then the erythrocyte lysate
erythrocyte lysate was collected and used to determine reduced glutathione (GSH) levels and catalase
was collected and used to determine reduced glutathione (GSH) levels and catalase (CAT) activity
(CAT) activity as previously described[69].The total protein concentration in the plasma was
as previously described [69].The total protein concentration in the plasma was determined by the
determined by the Bradford assay [70]. A BC-5000 Vet auto hematology analyzer (Mindray, North
Bradford assay [70]. A BC-5000 Vet auto hematology analyzer (Mindray, North America) was used to
America) was used to determine the hemoglobin concentration.
determine thefor
Briefly, hemoglobin
TAC analysis, concentration.
plasma samples were diluted in 0.01 M PBS at neutral pH (dilution
Briefly, for TAC analysis,
factor = 1:25). The diluted samples plasma samples
were mixed withwere0.01diluted
M DPPH insolution
0.01 M in PBS1:1at neutral
ratio pH (dilution
and incubated
factor = 1:25). The diluted samples were mixed with 0.01 M DPPH solution
for half-hour time in a dark chamber at room temperature (RT). After the incubation time, the samples in 1:1 ratio and incubated
for half-hour time in a dark chamber at room temperature (RT). After the
were centrifugated for 3 min at 20,000× g at 4°C(Eppendorf 5417 R centrifuge), and the supernatant incubation time, the samples
were centrifugated for 3 min at 20,000× ◦
g at 4 TheC(Eppendorf 5417 R centrifuge),
was collected for spectrophotometric analysis. samples absorbance at 517 nm and (A250)thewas
supernatant
read
was
usingcollected
a Hitachifor spectrophotometric
UV-VIS spectrophotometer, analysis.
and TAC Thewas samples absorbance
expressed as mmolat of 517 nm (A250)
reduced was read
DPPH using
using a Hitachi
the molar UV-VIS
extinction spectrophotometer,
coefficient of DPPH (11,500 andM TAC
−1·cm was
−1). expressed as mmol of reduced DPPH using

TBARS
the molar assay was
extinction performed
coefficient of as follows:
DPPH the 35%
(11,500 −1
M TCA ·cm−1was ). mixed with 0.2 MTris-CL at neutral
pH in 1:1 ratio.
TBARS assayOnewaspart of the plasma
performed sample
as follows: thewas
35%added TCA wasto 9 mixed
parts ofwith TCA/TRIS-Cl
0.2 MTris-CL mixture and pH
at neutral
incubated at RT for 10 min. After incubation time an equal volume of
in 1:1 ratio. One part of the plasma sample was added to 9 parts of TCA/TRIS-Cl mixture and incubated2 M sodium sulfate in 0.05
atMthiobarbituric
RT for 10 min. acidAfter (TBA) mixturetime
incubation wasan added
equalinvolume
the sample, of 2 Mheated
sodium at 95C
sulfateforin450.05
minMthiobarbituric
on a water
bath(TBA)
acid and then cooledwas
mixture on ice for 5 min.
added in theTCA (70%) heated
sample, was added at 95in the
◦ sample
C for 45 min andon mixed using
a water a vortex.
bath and then
After that, the samples were centrifuged at 15,000× g for 3 min (Eppendorf
cooled on ice for 5 min. TCA (70%) was added in the sample and mixed using a vortex. After that, 5417 R centrifuge). The the
supernatant was collected and analyzed by spectrophotometry at 532
samples were centrifuged at 15,000× g for 3 min (Eppendorf 5417 R centrifuge). The supernatant was nm. The TBARS level was
expressed using the molar extinction coefficient of MDA-TBA abduct (155 mM−1 cm−1).
collected and analyzed by spectrophotometry at 532 nm. The TBARS level was expressed using the
PCARB assay was performed as follows: plasma sample and 20% TCA were mixed in 1:1 ratio
molar extinction coefficient of MDA-TBA abduct (155 mM−1 cm−1 ).
and incubated on ice for 15 min. After incubation time, the samples were separated by centrifugation
at 15,000× g for 5 min at 4°C(Eppendorf 5417 R centrifuge), and the supernatant was removed. A
volume of 0.5 mL of 0.01 M of DNPH solution was added on the pellet. For the blank, we added 2.5
N HCL. After that, an incubation step was performed for 60 min in a dark chamber with intermittent
vortexing. After the incubation step, the samples were centrifuged at 15,000× g for 5 min at
Int. J. Mol. Sci. 2020, 21, 1233 12 of 17

PCARB assay was performed as follows: plasma sample and 20% TCA were mixed in 1:1 ratio
and incubated on ice for 15 min. After incubation time, the samples were separated by centrifugation at
15,000× g for 5 min at 4◦ C(Eppendorf 5417 R centrifuge), and the supernatant was removed. A volume
of 0.5 mL of 0.01 M of DNPH solution was added on the pellet. For the blank, we added 2.5 N HCL.
After that, an incubation step was performed for 60 min in a dark chamber with intermittent vortexing.
After the incubation step, the samples were centrifuged at 15,000× g for 5 min at 4◦ C(Eppendorf 5417
R centrifuge). After separation by centrifugation, the supernatant was removed, and the pellet was
washed three times with ethanol-ethylacetate solution in equal parts (v/v). The pellet was then solved
in 5 M urea at pH = 2.3 followed by vortex and incubation at 37 ◦ C for 15 min. After incubation
time, the samples were centrifuged at 15,000× g for 3 min at 4◦ C(Eppendorf 5417 R centrifuge) and
the supernatant was collected and analyzed by spectrophotometry at 375 nm. The PCARB content
from total protein concentration (analyzed by the Bradford method) was calculated using the molar
extinction coefficient of DNPH (22 mM–1 cm –1 ).
In order to assess the GSH level and CAT activity, we performed the erythrocyte lysate using an
equal volume of distillate water, mix by inversion and centrifuged for 15 min at 4020× g using a 4◦ C
centrifuge (Eppendorf 5417 R).
GSH level was assessed in erythrocyte lysate as follows: the erythrocyte lysate was treated with
5% TCA (v/v), vortex and separated by centrifugation at 28,000× g for 5 min at 4◦ C (Eppendorf 5417 R
centrifuge). In the sample, a mixture of 0.01 M DTNB in 0.07 M PBS at alkaline pH (pH = 8) in 1:50
ratio (v/v) was added and incubated in a dark chamber at RT for 45 min. After incubation time, the
samples were analyzed by spectrophotometry at 412 nm using a UV-VIS spectrophotometer (Hitachi).
The GSH concentration was calculated using a GSH standard curve.
For CAT activity analysis, we used 4 microL of 1:10 dilution of erythrocyte lysate sample in 3 ml
of 0.07 M PBS at neutral pH. The mixture was incubated at 37 ◦ C for 10 min, and the changes in sample
absorbance at 240 nm after peroxide addition wereread using a UV-VIS spectrophotometer (Beckman
UV-VIS). The CAT activity was calculated based on the molar extinction coefficient of peroxide and
expressed as Unit per mg of hemoglobin (U/mgHb).

4.7. Statistical Analysis

All statistical analyses were performed using STATA 13 (StataCorp. 2013. Stata Statistical Software:
Release 13. College Station, TX: StataCorp LP). Continuous data were expressed as arithmetic mean
(Average) ± standard deviation of the mean. To determine the difference between the groups in
normally distributed data, we used a one-way analysis of variance (ANOVA) and Tukey’s post hoc tests.
In the case of non-normally distributed data, we used Kruskal–Wallis and post-hoc Mann–Whitney
tests with Holm–Sidak adjustment. A value of p<0.05 was considered statistically significant.

5. Conclusions
EG-AgNPs manifest antioxidant effects that protect against oxidative stress in subacute exposure,
while PVP-EG-AgNPs manifest pro-oxidant effects at the same doses and in the same administration
regimen. The EG-AgNPs protect against oxidative stress by increasing TAC and CAT levels and
decreasing TBARS and PROTC levels. The mechanism of PVP-EG-AgNPs induction of oxidative
stress is mediated by the induction of protein oxidation and decreased GSH levels. The different
mechanisms of EG-AgNPs and PVP-EG-AgNPs on antioxidant-/pro-oxidant balance can be explained
by the influence of the coating agent used for the preparation of the nanoparticles in the formation and
composition of protein corona that influence the pathophysiology of the nanoparticles in the organism.
Further studies should be carried out in order to evaluate the chronic effects of exposure to these types
of NPsand also the beneficial effects of synthesized PVP-EG-AgNPs in cancer treatment based on their
properties to induce ROS.
Int. J. Mol. Sci. 2020, 21, 1233 13 of 17

Author Contributions: A.O.D., D.C., A.M.B., O.Z. contributed equally in writing the manuscript and substantially
contributed to study design, data acquisition, including data analysis and interpretation. A.O.D., D.C., M.M.B.P.
and L.M. coordinated conception of the study. O.Z., G.D.M., C.T.S. and E.L.P. used statistical software for
the data interpretation. A.E.S., A.C.B., B.S.V. and A.M.G. contributed to writing the manuscript. A.O.D.,
D.C. and L.M. supervised the whole process. All authors have read and agreed to the published version of
the manuscript.FundingThis work was funded by a grant from the Romanian National Authority for Scientific
Research and Innovation, UEFISCDI, project number 45PCCDI/2018–“Bioactive nanostructures for innovative
therapeutic strategies”.
Conflicts of Interest: The authors declare no conflict of interest.

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