Enhanced photocatalysis and anticancer activity of green hydrothermal synthesized Ag?TiO2 nanoparticles

Article abstract of DOI:10.1016/j.jphotobiol.2019.111636

Titanium dioxide (TiO₂) nanoparticles (NPs) have been doped with varying amounts (0.005, 0.010 and 0.015 M) of silver nanoparticles (Ag NPs) using hydrothermal method. Further, in this work, a green approach was followed for the formation of Ag?TiO₂ NPs using Aloe vera gel as a capping and reducing agent. The structural property confirmed the presence of anatase phase TiO₂. Increased peak intensity was observed while increasing the Ag concentration. Further, the morphological and optical properties have been studied, which confirmed the effective photocatalytic behavior of the prepared Ag?TiO₂ NPs. The photocatalytic performance of Ag?TiO₂ has been considered for the degradation of picric acid in the visible light region. The concentration at 0.010 M of the prepared Ag?TiO₂ has achieved higher photocatalytic performance within 50 min, which could be attributed to its morphological behavior. Similarly, anticancer activity against lung cancer cell lines (A549) was also determined. The Ag?TiO₂ NPs generated a large quantity of reactive oxygen species (ROS), resulting in complete cancer cell growth suppression after their systemic in vitro administration. Ag?TiO₂ NPs was adsorbed visible light that leads to an enhanced anticancer sensitivity by killing and inhibiting cancer cell reproduction through cell viability assay test. It was clear that 0.015 M of Ag?TiO₂ NPs were highly effective against human lung cancer cell lines and showed increased production of ROS in cancer cell lines due to the medicinal behavior of the Aloe vera gel.

Full text of DOI:10.1016/j.jphotobiol.2019.111636

JournalofPhotochemistry&Photobiology,B:Biology202(2020)111636
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Enhanced photocatalysis and anticancer activity of green hydrothermal
synthesized Ag@TiO2 nanoparticles
D. Hariharana, P. Thangamuniyandib, A. Jegatha Christyc, R. Vasantharajad, P. Selvakumare,
⁎
S. Sagadevanf, A. Pugazhendhig, L.C. Nehrua,
a Department of Medical Physics, School of Physics, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India
b School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India
c PG & Research Center of Physics, Jayaraj Annapackiam College for Women, Periyakulam, Tamil Nadu, India
d Centre for Ocean Research, Sathyabama Institute of Science and Technology, Chennai 600 119, Tamil Nadu, India
e Department of Physics, Coimbatore Institute of Technology, Coimbatore 641 014, Tamil Nadu, India
f Nanotechnology & Catalysis research Center, Institute of Advanced Studies, University of Malaya, Malaysia
g Innovative Green Product Synthesis and Renewable Environment Development Research Group, Faculty of Environment and Labour Safety, Ton Duc Thang University, Ho
Chi Minh City, Viet Nam
A R T I C L E I N F O
A B S T R A C T
Keywords:
Titanium dioxide (TiO2) nanoparticles (NPs) have been doped with varying amounts (0.005, 0.010 and 0.015 M)
of silver nanoparticles (Ag NPs) using hydrothermal method. Further, in this work, a green approach was fol-
lowed for the formation of Ag@TiO2 NPs using Aloe vera gel as a capping and reducing agent. The structural
property confirmed the presence of anatase phase TiO2. Increased peak intensity was observed while increasing
the Ag concentration. Further, the morphological and optical properties have been studied, which confirmed the
effective photocatalytic behavior of the prepared Ag@TiO2 NPs. The photocatalytic performance of Ag@TiO2
has been considered for the degradation of picric acid in the visible light region. The concentration at 0.010 M of
the prepared Ag@TiO2 has achieved higher photocatalytic performance within 50 min, which could be attrib-
uted to its morphological behavior. Similarly, anticancer activity against lung cancer cell lines (A549) was also
determined. The Ag@TiO2 NPs generated a large quantity of reactive oxygen species (ROS), resulting in com-
plete cancer cell growth suppression after their systemic in vitro administration. Ag@TiO2 NPs was adsorbed
visible light that leads to an enhanced anticancer sensitivity by killing and inhibiting cancer cell reproduction
through cell viability assay test. It was clear that 0.015 M of Ag@TiO2 NPs were highly effective against human
lung cancer cell lines and showed increased production of ROS in cancer cell lines due to the medicinal behavior
of the Aloe vera gel.
Hydrothermal synthesis
Photocatalyst
Aloe vera
Picric acid
ROS
Cancer cell lines-A549
1. Introduction
improves quantum yield but also generates excess free radicals that lead
to enhanced photocatalytic activity. TiO2 NPs generate free radicals
Generally, TiO2 photocatalytic materials play potential roles in
water treatment, catalysis, lithium-ion batteries, and sensors, antic-
ancer and antibacterial applications [1,2]. TiO2 absorbs only UV light
that leads to low quantum yield [3]. Noble metals loaded on the surface
of TiO2 enable Visible light absorption-due to Schottky barrier effect.
Among the noble metals, silver is easily available and has high work
function, antibacterial property and more importantly, its SPR effect at
the desired wavelength [4]. In Ag@TiO2 formation, Ag (4d) promotes
effective photo generated electron hole separation, which leads to
narrow band gap that promotes visible light absorbance [5].
such as superoxide radical (O2−·), hydroxyl radical (·OH), and singlet
oxygen (1O2). Meanwhile, Picric acid (PA) is a well-known strong nitro
aromatic compound and its salts are used as explosives even greater
than many other polynitrated aromatic compounds such as trini-
trotoluene [6]. Furthermore, the acquaintance to picric acid (PA) leads
to adverse effects to our environment. In addition, PA also shows ser-
ious effects on human health such as eye and skin irritations, causes
respiratory problems and affects the immune system [7]. Above all, the
biodegradation products of PA are of concern due to their oncogenic
properties, which allow them to easily affect our human society [8].
Though a large number of catalytic materials are reported on TiO2, a
Visible light absorbance of TiO2 NPs not only promotes and
⁎ Corresponding author.
E-mail addresses: arivalagan.pugazhendhi@tdtu.edu.vn (A. Pugazhendhi), lcnehru@bdu.ac.in (L.C. Nehru).
https://doi.org/10.1016/j.jphotobiol.2019.111636
Received 24 June 2019; Received in revised form 6 September 2019; Accepted 19 September 2019
Availableonline12November2019
1011-1344/©2019PublishedbyElsevierB.V.

D. Hariharan, et al.
JournalofPhotochemistry&Photobiology,B:Biology202(2020)111636
simple and practical approach to the degradation of PA still remains a
challenge. In this context, the present study has focused on the effective
photocatalytic degradation of such adverse PA using Ag@TiO2 NPs.
In cell line studies, TiO2 NPs induce the cell lines and ROS is pro-
duced, which damages DNA, leading to apoptosis, and necrosis. A549
cell line is an adenoma lung cancer cell line, and it is used to investigate
the impact of NPs. Swarnalatha and Anjaneyulu, have reported picric
acid degradation in aqueous TiO2 suspension under UV light [9]. Ta-
naka et al. reported mono-, di- and trinitro phenol degradation in
aqueous TiO2 suspension under UV light irradiation [10]. The efficiency
of picric acid degradation process in the visible light region has also
been reported Hariharan et al. [11]. Kim et al. have reported that ZnO
had shown an enhanced anticancer activity on A549 cell lines com-
pared to TiO2, Al2O3, and CeO2 [12]. There is no report on green hy-
drothermal synthesized Ag@TiO2 nanostructures with anticancer ac-
tivity against A549 cell lines.
spectrometer with MgKα X-rays as the excitation source. Powder X-ray
diffraction (XRD) measurements of the samples have been performed
with a Philips PW3040/60, USA X-ray diffractometer using CuKα ra-
diation at a scanning rate of 0.06° s−1. Raman spectral studies were
carried out using a laser confocal Raman microscope (Renishaw, UK,
Model: Invia). The presence of functional groups was identified using
Fourier transform infrared (FTIR) spectrum in the range of
4000–500 cm−1 with an Alpha II Bruker FTIR spectrometer, USA.
2.3. Preparation of Aloe vera Gel
The preparation of Aloe vera gel was performed following the
method of Ali et al. [16]. Herein, the Aloe vera leaf was rinsed twice
using double distilled water in order to remove all the debris and visible
unwanted particles from the surface. Then, the leaf was carefully peeled
off using a sharp knife and peel was discarded. The leaf was slit long-
itudinally, and the gel was scraped off from the leaf and then, trans-
ferred into a sterile beaker, using a sharp-edged spoon. Then, 10 ml of
the gel was added to 100 ml distilled water and boiled for 2 h at 90 °C.
The aqueous gel was filtered using Whatman filter paper. The gel was
stored at 4 °C for further studies.
In general, plant biomolecules act as capping and reducing agents
for the synthesis of NPs [13]. Herein, Aloe vera gel could be generally
used as an antioxidant due to the presence of different types of vitamins
such as A, C and E [14]. Moreover, the gel extracted from the peel is
able to retort and heal burns and wounds and can be a UV protecting
material. Further, Aloe vera gel has been used as a capping and reducing
agent for NPs, which aids to green synthesize NPs with high crystalline
and optical properties compared to chemical synthesis [15]. With the
medicinal importance of the Aloe vera gel mentioned above, the present
study has focused on the synthesis, characterization and aimed to study
the anticancer activity of the extracted Ag@TiO2 NPs.
2.4. Hydrothermal Synthesis of TiO2 NPs
TiO2 nanostructures were prepared by the green hydrothermal
method with slight modifications of the procedure reported in literature
[11]. Ten milliliters of titanium tetra-isopropoxide (TTIP) was taken in
a 60 ml Teflon beaker, 20 ml of distilled water was then added, and the
above solution was kept in a stainless steel autoclave. It was then kept
in a muffle furnace and heated at 180 °C for 24 h. The obtained
homogeneous solution was kept in the autoclave and dried at 120 °C for
2 h. The final product was calcinated at 500 °C for 5 h.
In this research, the green hydrothermal synthesized Ag@TiO2 NPs
have been reported to be effective for visible light active picric acid
degradation process and have shown anticancer activity against A549
cell lines. To the best of knowledge of the authors, this is the first report
on using Ag@TiO2 NPs for degradation of picric Acid. It is clear that
Ag@TiO2 NPs are highly effective against human lung cancer cell lines
due to the medicinal properties of Aloe Vera gel and increased pro-
duction of ROS in cancer cell lines. Herein, the present effective en-
vironmentally safe biogenic green synthesis of Ag@TiO2 NPs would be
an appropriate way to obtain highly dispersed silver photocatalytic
behavior over PA degradation activity and the as-prepared NPs also can
be used in effective anticancer therapy.
2.5. Hydrothermal Synthesis of Ag@TiO2 NPs
As per our previous report [17], different concentrations of
(0.005 M, 0.010 M and 0.015 M) silver nitrate (AgNO3) were added
with 0.1 M Titanium (IV) isopropoxide, 5 ml of Aloe Vera gel and 100 ml
of water. The above solution was stirred for 1 h then, it was kept in a
stainless steel autoclave, which was placed on a muffle furnace and
heated for one day at 180 °C. The obtained product was dried at 120 °C
for 2 h. Finally, the obtained powder was calcinated at 500 °C for 5 h.
2. Materials and Methods
2.1. Materials
2.6. Photocatalytic Experiments
Titanium (IV) isopropoxide (99.8%), silver nitrate (≥99%), 2,4,6-
trinitrophenol (C6H3N3O7, 98%) and (3-(4,5-dimethylthiazol-2-yl)-
2,5Diphenyl tetrazolium Bromide (≥99%) have been procured from
Sigma–Aldrich and used as such. Double Distilled (DD) water has been
used to prepare the solutions. All the appliances have been rinsed with
aquaregia and washed with DD water before use. Aloe barbadensis (Aloe
Vera) plants have been collected at the Bharathidasan University
campus, Bharathidasan University, Trichy, Tamil Nadu, India.
Photocatalytic experiments were carried out according to the pre-
viously described method [18]. Picric acid degradation by the prepared
Ag@TiO2 nanostructures was evaluated. In each experiment, Ag@TiO2
catalyst (20 mg) was incorporated in 100 ml of picric acid solution
(500 mg/L). Then, the above solution was stirred in a dark place for
30 min for absorption-desorption equilibrium. The experiments were
carried out in visible light irradiation on picric acid suspension using a
high-pressure mercury lamp (543 nm). Picric acid suspension was
drawn every 5 min and evaluated using UV–Visible spectrophotometer
at λmax = 353 nm.
2.2. Characterization Techniques
The ultraviolet-visible (UV–Vis) spectrum was recorded using, a UV-
160 UV − Vis spectrophotometer (Shimazu Co., Japan). High-
Resolution Transmission Electron Microscopy (HRTEM) Selected Area
Electron Diffraction (SAED) pattern was collected at 200 kV using a
JEM-2100F, JEOL, Japan. Energy Dispersive X-ray Spectrometry (EDS)
has been performed with TEM. The samples utilized for TEM mea-
surements have been prepared by dispersing the products in ethanol as
well as placing several drops of the suspension on holey carbon films
supported by copper grids. Further evidence for the composition of the
product has been inferred from X-ray Photoelectron Spectroscopy
(XPS), through an ESCA Lab MKII, UK. X-ray photoelectron
2.7. Cell Culture and MTT Cell Viability Assay
Assay was carried out according to the previously described method
of Vasantharaja et al. [19]. Human lung carcinoma cells, which were
procured from the national center for cell sciences, Pune, India, were
maintained in DMEM medium containing 10% FBS, 100 mg/ml strep-
tomycin, 100 IU/ml penicillin and 2 mM glutamine. The cultures were
maintained at 37 °C with 5% CO2 in a humidified CO2 incubator. The
Ag@TiO2 and TiO2 NPs were evaluated for cytotoxic activities against
human lung carcinoma (A549) cell lines. The cultural A549 cells were
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D. Hariharan, et al.
JournalofPhotochemistry&Photobiology,B:Biology202(2020)111636
seeded separately on 96 well plates at a concentration of 1 × 104 cells/
well. The cells were subjected to different concentrations of green
synthesized Ag@TiO2 and TiO2 (0 to 200 μg/ml). After 24 h, 100 μl of
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
was added and incubated at 37 °C for 4 h in a CO2 incubator. After
incubation, the purple colored formazan was dissolved by adding
100 μL of Dimethyl sulfoxide. After 30 min, the optical density was
measured at 570 nm by using an ELISA multiwell plate reader for de-
termining IC50 values. All the tests were performed in triplicates to
avoid any experimental error.
smooth compared to Ag@TiO2 NPs. After depositing different Ag con-
centrations on TiO2 NPs, the surface of TiO2 NPs was changed from
Fig. 1(C), it could be observed that when Ag concentrations have been
changed, morphologies of Ag@TiO2 NPs have also changed. Due to
hydrothermal treatment, the rod shaped TiO2 NPs and small spherical
Ag NPs were formed as shown in Fig. 1 (B & D). The doping con-
centration and the hydrothermal method of preparation could have
been the two major factors influencing the morphological changes ob-
served [23–25]. The calculated total size of Ag NPs was 38 nm and the
size of TiO2 NPs was 57 nm. The SAED pattern lattice springs clearly
showed the anatase phase that was matched with (101) crystallography
plane. In Ag@TiO2 nanostructures, the percentage of Ag was 1.77%
TiO2 was 69.05% and O was 29.24%, respectively as denoted by EDAX
spectrum shown in supplementary file (Fig. S2).
2.8. Acridine Orange/Ethidium Bromide (AO/EtBr) Staining
AO/EB double staining assay was used to determine the ability of
Ag@TiO2 and TiO2 NPs to induce apoptosis (A549 cells). The staining
assay was carried out according to the previously described method
[20]. A549 cells were seeded separately in six-well plates with the re-
spective IC50 concentrations of Ag@TiO2 and TiO2 NPs and were in-
cubated for 24 h. Fluorescent dyes viz., Ethidium bromide (100 μg/ml)
and Acridine orange (100 μg/ml) solutions were mixed with respective
cells. The cells were incubated at 37 °C for 30 min in dark. Later, AO/EB
staining was observed through a fluorescence microscope. The me-
chanism of AO/EB double staining assay presents that the cells con-
taining normal nuclear chromatin portray green nuclear staining (AO is
taken up by viable cells). Apoptotic cells, which contained orange to red
nuclei with condensed chromatin (EO is taken up by non-viable cells)
were evaluated by fluorescence microscope at 20× magnification.
3.3. XPS Studies of TiO2 and Ag@TiO2 NPs
X-ray photoelectron spectroscopy was used to analyze the chemical
state and elemental composition. Fig. 2 (A) (i-iv) shows the XPS spectra
of TiO2 and Ag@TiO2. Ti, C, Ag, and O and they are presented in green
hydrothermal synthesized Ag@TiO2 (Table. S1, S2). Ti 2p spectrum
shows peaks at 458.4 eV (Ti 2p3/2) and 464.2 eV (Ti 2p1/2). The position
of peak was 5.2 eV indicating the Ti4+ [26] oxidation state for TiO2
nanostructure. The peak position for C1s in the Ag@TiO2 samples could
be decomposed into three chemically distinct components present in
the biomolecules (Fig. 2(B)- ii). Besides the C1s peak at 284.5eVBE
(major peak) which corresponded to the carbon atoms within amino
acids, vitamins, lignin, anthraquinones, enzymes, polysaccharides, sal-
icylic acids, minerals, monosaccharides, saponins and sterols, two other
peaks at 284.4 and 288.2 eV BE have also been observed. The higher BE
(binding energy) peak at 288.3 eV was attributed to the electron
emission from carbons in carbonyl groups [27] (Carbonyl carbons of
the polysaccharides or proteins-enzymes) and the lower peak at
284.4 eV BE would be most likely from carbons to the carbonyl carbon.
Compared to TiO2 (Fig. 2 (A) - i), the peaks of Ag@TiO2 shifted to
higher binding energy. It confirms strong interaction between Ag and
TiO2 nanostructures. Using curve fitting program for Ag@TiO2 nanos-
tructures, Ti2P3/2 peaks were further divided into two peaks, and they
were present at 459.9 eV and 458.2 eV, caused by Ti4+ and Ti3+ spe-
cies, respectively. Evidently, the presence of Ti3+ species might be the
reason for visible light absorption and enhanced photocatalytic activity
in the visible light region [11].
2.9. Dichloro Dihydro Fluorescein Diacetate (DCFH-DA) Assay
The intracellular ROS generation by TiO2 and Ag@TiO2NPs for
A549 cells was detected by DCFH-DA staining. A549 cells were seeded
in six-well plates and treated with TiO2 and Ag@TiO2 NPs. After 24 h of
incubation, the cells were rinsed with 1× cold PBS and stained with
DCFH-DA (10 μg/10 μL) in dark condition for 30 min. Finally, the ROS
level was examined by fluorescence microscope (Floid cell imaging
station, Life technologies, U.S.A). The assay was carried out according
to the previously described method and in triplicates [21].
3. Results and Discussion
3.1. FT-IR-Analysis of Plant Leaf Extract
XPS spectra of O1s TiO2 NPs and Ag@TiO2NPs are presented in Fig.
(2(A) - iii) and B(iii). O1s spectra were further fitted for TiO2 and
Ag@TiO2 nanostructures, and they indicated (OTi-O at 529.6 eV for TiO2
and Ag@TiO2), surface silver groups (OO-Hat 531.7 eV and 531.4 eV for
TiO2 and Ag@TiO2) [28], and adsorbed O2 (at 531.7 eV and 533 eV for
TiO2 and Ag@TiO2) [29]. In Ag 3d spectrum, two peaks were observed
for Ag 3d5/2 (367.5 eV) and Ag3d3/2 (373.5 eV) at low binding energies
compared to bulk Ag (368.3 eV for Ag 3d 5/2, and 374.3 eV for Ag 3d3/2
[30], due to an electron moving from Ag to TiO2 surface. Ag3d5/2 in-
dicated two peaks and they were Ago (367.3 eV) and Ag2O (368.5 eV).
After visible light irradiation, Ag ions were transferred to Ag2O, when
Ago was on the surface. It confirmed that Ag was strongly oxidized,
during visible light exposure.
FTIR spectrum of 500 °C calcinated Aloe vera gel (Supplementary
file (Fig. S1)). Strong IR bands have been observed at 3459, 2085, 1634,
1550, 1409 and 662 cm−1. The spectrum for plant gel extract exhibited
intense and distinct absorption bands at 3459 cm−1, which corre-
sponded to NeH stretching. The bands at 2085 cm−1 were due to
N=C=S bond stretching, and 1550 cm−1 corresponded to NeO bond
stretching. The low band at 662 cm−1 corresponded to C–X stretching
(bending region). Generally, plant gel extract is known to contain
biomolecules such as lignin, vitamins, amino acids, enzymes, anthrao-
quinones, polysaccharides, salicylic acids, minerals, monosaccharide,
saponins, and sterols in different extracts of Aloe Vera aqueous gel [22].
The peak at 1409 cm−1was assigned to the COO– symmetric stretch
from proteins with carboxyl side groups in the amino acid residues.
Further, the peak that appeared at 1634 cm−1 corresponded to the
stretching modes of –C=O group. The above biomolecules might have
contributed to the reduction of Ag+ ions.
3.4. Structural Studies of TiO2 and Ag@TiO2 NPs
TiO2 and Ag NPs anchored TiO2 NPs were analyzed for crystallinity.
Fig. 3 (A) shows that diffraction peaks were not associated with crys-
talline TiO2. It demonstrated that TiO2 was amorphous in both the as-
deposited intrinsic and NPs samples. After annealing at 500 °C, the
diffraction peaks were observed at 2θ = 25.6°, 38.0°, 48.4°, 54.2°,
55.3°, 63.0°, and 69.0°, respectively to the (101), (112), (200), (105),
(211), (204) and (220), crystal planes of the TiO2 anatase phase (JCPDS
file No.21–1272), which are presented in Fig. 3 (A) (b-d). All the
3.2. HRTEM Analysis of TiO2 and Ag@TiO2
The morphology properties of the pure TiO2 NPs and Ag@TiO2
nanostructures have been investigated by HRTEM. Fig. 1 (A) shows the
HRTEM image of TiO2 NPs and Fig. 1 (B–D) shows the Ag@TiO2 na-
nostructures. In Fig. 1 (A), surface of the nanostructure was very
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D. Hariharan, et al.
JournalofPhotochemistry&Photobiology,B:Biology202(2020)111636
Fig. 1. TEM images: (A) Pure TiO2; (B) Ag@TiO2–0.005 M; (C) Ag@TiO2–0.010 M; (D) Ag@TiO2–0.015 M; (E) HRTEM image of Ag@TiO2 nanostructures (Inset:
selected area electron diffraction (SAED) from a single Ag nanocrystal).
Ag@TiO2 samples focused on the usual pure anatase phase (indicated A
in XRD) and Rutile phase (indicated R in XRD) with similar peak in-
tensities and shapes (like original TiO2). Even at the highest con-
centration (0.015 M) of the AgNO3 solution utilized for the deposition
of Ag, new diffraction peaks were not related to Ag species, which was
because of the least amount of Ag and the amorphous state of Ag [31].
In addition, no changes were observed in the positions of diffraction
peaks. It showed that Ag has been deposited on TiO2 surface by
Ag@TiO2 formation, which was supported by TEM and XPS observa-
tions. They indicated that parts of Ag and TiO2were incorporated with
the amorphous states, whereas a part of Ag aggregated into small
crystals in the as-deposited samples. The peaks became more intense as
the percentage of Ag increased. Further, the diffraction peaks of silver
were not observed. It indicated that in the as-deposited samples, there
was a very small amount of silver, which was below the instrument's
detection limit.
decreased with an increasing amount of Ag. It denoted that there was
an interaction between the Ag and TiO2 NPs in order to influence the
resonance of Raman effect for TiO2 NPs.
3.6. UV–Visible Spectra of TiO2 and Ag@TiO2 NPs
The UV–Vis absorption-diffusion reflectance spectra of pure TiO2
NPs and different concentrations of Ag in Ag@TiO2 nanostructures are
depicted in supplementary file (Fig. S3). The pure TiO2 NPs showed the
typical absorption of anatase with an intense transition in the UV re-
gion. There was no absorption in the visible light region whereas all the
Ag@TiO2 NPs presented prominent absorption in the region of
400–600 nm, indicating that the absorption of Ag@TiO2 has sig-
nificantly been red-shifted to the visible region due to the loading of the
Ag nanocrystals on the surface of theTiO2 NPs. It was due to the pro-
motion of the electron from the valence band to the conduction band.
The Ag@TiO2 NPs clearly showed the characteristic absorption of TiO2
NPs in the UV region, which could be attributed to the Ago Surface
Plasmon Resonance (SPR) with the TiO2 inter-band transition at
λ < 480 nm. Many of the factors such as quantity, particle size, dis-
persion and morphology of Ag NPs, can influence the SPR effect in-
tensity. Meanwhile, a decrease in the band gap values could be ob-
served for Ag@TiO2 photo catalysts. The Ag@TiO2 (AgNO3 0.015 M)
had a strong absorption band at around 470 nm (magnet curve) and the
observed result correlated with the previous report of Yu et al. on the
absorption spectra of Ag doped TiO2 [34]. It was also evident that the
intensity of the Ag@TiO2 NPs was much higher than that of the other
two Ag@TiO2 (0.005, 0.010 M) samples. It meant that the nanos-
tructures had a larger silver distribution. This decrease happened be-
cause of the Ag NPs that have introduced localized energy levels in the
band gap of TiO2 NPs. The electrons could be excited with low energy
from the valence band (VB) to these levels rather than to the conduction
band (CB) of the semiconductor. It should be noted that the nanos-
tructures of Ag@TiO2 with absorption in the visible region may involve
in an increased activity of photo catalysis [16].
3.5. Raman Spectra of TiO2 and Ag@TiO2 NPs
Raman spectroscopy has been further employed in the character-
ization of the crystalline structure of TiO2 NPs, and the results are
shown in Fig. 3(B). TiO2 anatase structure was characterized by the
tetragonal space group of I41/amd. TiO2 NPs contained twelve atoms
per unit cell with lattice parameters a = 3.784 Å and c = 9.514 Å.
Anatase contained six Raman active modes (A1g + B1g + 4Eg) and they
were allowed according to the factor group analysis. Ohsaka et al. have
reported the Raman spectrum of an anatase single crystal [32]. It ex-
plained that the six allowed modes appeared at 148 cm−1 (Eg),
199 cm−1 (Eg), 392 cm−1 (B1g), 445 cm−1 (Eg), 517 cm−1 (A1g), and
635 cm−1 (Eg), respectively. The Raman spectrum excited at 488 nm
for the three Ag@TiO2 samples was compared with the pure TiO2 NPs.
The prime Raman bands of all the samples were attributed to the
characteristic anatase phase of TiO2 NPs, dominated by the Raman
spectra of the Ag@TiO2 nanostructures. The observed result well mat-
ched with the previous report of Maury-Ramirez [33] that the prepared
Ag@TiO2 nanostructures confirmed the absence of signals related to Ag
nanocrystals, which could be attributed to a relatively low content of
Ag loaded onto the TiO2 backbone. Here, the most interesting phe-
nomenon of the spectra was that the intensity of the TiO2 related bands
3.7. Photocatalytic Studies of TiO2 and Ag@TiO2 NPs
Shvadchina et al. have reported the photocatalytic degradation of
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JournalofPhotochemistry&Photobiology,B:Biology202(2020)111636
Fig. 2. XPS spectra: (A) TiO2 samples: (i) Ti 2p; (ii) C1s; (iii) O 1 and (B) Ag@TiO2 samples: (i) Ti 2p; (ii) C1s; (iii) O 1 s; (iv) Ag 3d.
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D. Hariharan, et al.
JournalofPhotochemistry&Photobiology,B:Biology202(2020)111636
degradation can be summarized as follows.
Ag@TiO2 (0.010M) > Ag@TiO2 (0.015M) > Ag@TiO2 (0.005M) > TiO2
Photocatalytic charge separation process under visible light is listed
in Eqs. (1)–(7) as given below;
·
Ag@TiO2 (e ) + O2 Ag@TiO2 + O2
(1)
·
·
O2 + H(+) HO2
(2)
HO2 + H(+)
e
H2O2
(3)
(4)
·
·
·
H2O2 + O2
OH + OH + O2
hv
H2O2
2OH
(5)
(6)
(7)
·
Ti3+ + H2O2 Ti4+ + OH + OH
OH (or O2 ·) + PA degraded or mineralized products
3.8. Determination of Photodegradation Products
The total molecular picric acid was being degraded for 0, 10, 20, 30,
40 and 50 min, respectively. As seen in Fig. 5 (a), for ‘0’ min, there were
two small peaks of m/z, 113, and 181 except for a strong peak at m/z
91, 228 corresponding to phenol and PA. The peak of m/z 181 could be
attributed to the loss of one NO2 from PA. The LC-MS spectrum of an
ion at m/z 181 yielded the fragment ions corresponding to [M-3H]+
.
Since these peaks existed in the samples before the visible light irra-
diation, they might be attributed to the limited degradation of PA in
room light during the LC-MS operating procedures.
After 30 min irradiation, it was found that two new peaks (m/z 194
and 175) were clearly observed (shown in Fig. 5 (b)). The peak of m/z
194 always existed during the photo degradation of PA for 30 min;
perhaps it belonged to the impurity in the solution. The parent molecule
with m/z 228 lost one nitro group corresponding to m/z 181 (Fig. 5 (c))
LC-MS peak of m/z 175 yielded the fragment ions corresponding to
[M + CH3OH + 4H]+. Additional peak appear m/z 157 yielded the
fragment ions corresponding to [M + CH3OH + H]+. Another frag-
ment peak of m/z 97 The compound of m/z 66 was the cyclopentadiene
product of picric acid.
Fig. 3. (A) XRD patterns: (a) Pure TiO2; (b) Ag@TiO2–0.005 M; (c)
Ag@TiO2–0.01 M; (d) Ag@TiO2–0.015 M; (B) Raman spectra of: (a) pure TiO2;
(b) Ag@TiO2–0.005 M; (c) Ag@TiO2–0.010 M; (d) Ag@TiO2–0.015 M.
PA using S-TiO2 doped with sulphur and pure TiO2. They have observed
the photocatalytic behaviors of both S doped and pure TiO2 samples in
the UV region, which were less effective at oxidation by picric acid
compared to that of the oxidation by fulvic acids and humic acid [36].
However, the fermi energy level of Ag is higher than TiO2, and hence,
the electron transfers from Ag to TiO2. During visible light irradiation
on the photocatalytic process, Ag and TiO2 form new fermi energy le-
vels and adjust the same value by Schottky barrier effect mechanism,
which [37,38] leads to a higher photocatalytic activity. Fig. 4, shows
the photocatalytic degradation of PA using pure TiO2 and 0.005,
0.015 M Ag@TiO2 NPs. Without using a catalyst, there was no de-
gradation spectrum in PA. Fig. 4 (b, c, and d) show the PA degradation
spectra within 100, 70, 60 min, respectively, when the pure TiO2,
0.005 M and 0.015 M Ag@TiO2 acted as catalysts. PA was not degraded
when the catalyst was not active during visible light irradiation as
shown in Fig. 4(a). A better result was achieved for 0.010 M Ag@TiO2
NPs with PA degradation within 50 min (Fig. 4 (e)). According to the
Schottky barrier effect, Ag concentration was increased, and the visible
light adsorption was increased. The increased visible light adsorption is
usually directly proportional to the enhanced photocatalytic activity.
This confirmed the increased concentration of Ag being directly pro-
portional to the increased photocatalytic activity. In the present study,
0.010 M Ag@TiO2 has increased photocatalytic activity compared to
0.015 M Ag@TiO2 NPs due to photocatalytic activity depending on the
shape of the NPs. The shape of 0.010 M Ag@TiO2 NPs was different
compared to 0.005 M and 0.015 M Ag@TiO2 as depicted by HRTEM.
Fig. 4(g) describes that acid is not degraded in acetic nature (pH. 1–7).
Hence, pH value was increased, and PA was degraded in basic condi-
tions (pH =10). The order of photocatalytic activity of picric acid
Another possible fragmentation of the LC-MS spectrum of an ion at
m/z 181 yielded the fragmentations corresponding to [M-3H]+. The
compound of m/z 228 could be attributed to degradation and it had lost
one nitro group from picric acid. The LC-MS spectrum of an ion at m/z
133 yielded the fragmentations corresponding to [M-6H]+ The peak at
m/z 97 yielded the fragmentations corresponding to [M]+ and me-
chanism for the final product of picric acid was cyclopentadiene [M-
C5H6]+ at m/z 66 given in Fig. 5 (d)).
3.9. Reactive Oxygen Species (ROS) Analysis of Ag@TiO2 and TiO2 NPs
The reactive species level has been determined by the DCFH-DA
stain for Ag@ TiO2 NPs and TiO2 NPs. Control cells showed (Fig. 6a)
low light emission of green fluorescence compared to TiO2 NPs
(Fig. 6b). Very bright light was observed for various concentrations of
Ag@TiO2 NPs (Fig. 6c–e). This was due to the non-fluorescent DCFH-
DA that penetrated the cell, and the intracellular oxidation converting
the fluorescent dye [39]. It confirmed that when ROS was increased the
bright light would also increase. The highest bright light (increased
ROS production) has been achieved for 0.015 M Ag@TiO2 NPs. This
was because of the ability of the production of free radical elements by
NPs and the induction of reactive and nonreactive free radical elements
in the cell cytoplasm. These were major crucial regulators or mediators
of cell cycle arrest and apoptosis induction. The increased concentra-
tion of silver nitrate for doping (0.005, 0.010, 0.015 M), showed dra-
matically increasing production of ROS in cellular metabolism. The
unbalanced metabolic rate is a real path leading to cell death [40].
6

D. Hariharan, et al.
JournalofPhotochemistry&Photobiology,B:Biology202(2020)111636
Fig. 4. Absorption spectra of PA (a) without catalyst; (b) Pure-TiO2, (c) Ag@TiO2–0.005 M (d) Ag@TiO2–0.015 M, (e) UV–vis absorption spectra for degradation of
picric acid Ag@TiO2 (0.010 M) kinetic linear simulation curves of PA photocatalytic degradation with (f) pH variation (g) Catalyst variation.
7

D. Hariharan, et al.
JournalofPhotochemistry&Photobiology,B:Biology202(2020)111636
Fig. 5. LC-MS spectrum of picric acid degradation at (a) 0 min (b) 30 min, (c) Picric acid degradation products; (d) Mechanism of the proposed degradation products
of picric acid.
8

D. Hariharan, et al.
JournalofPhotochemistry&Photobiology,B:Biology202(2020)111636
Fig. 6. ROS images for A549 cell lines using (a) Control; (b) TiO2; (c) 0.005 M of Ag@TiO2; (d) 0.010 M of Ag@TiO2; (e) 0.015 M of Ag@TiO2.
Fig. 7. Stain analysis image of A549 cell lines using (a) Control; (b) TiO2; (c) 0.005 M Ag@TiO2; (d) 0.010MAg@TiO2; (e) 0.015 M Ag@TiO2.
3.10. Cell Growth Inhibition of Ag@TiO2 and TiO2 NPs
through hydrothermal method using Aloe vera gel. Structural study
confirmed the presence of Ag@TiO2 NPs. The photocatalytic perfor-
mance of Ag@TiO2 has been considered for the degradation of picric
acid in the visible light region, which is due to their UV absorption
phenomena and Ti3+ oxidation state confirmed through XPS result.
0.010 M of the prepared Ag@TiO2 has achieved a higher photocatalytic
performance within 50 min, which attributed to the morphological
impacts observed. Compared to the other concentrations, 0.015 M of
Ag@TiO2 showed better optical property having high visible light ab-
sorption that led to an excess electron transfer to cell lines. This resulted
in the increased production of ROS in the cell lines increasing the cell
death, confirming the enhanced anticancer activity. The simple and
biogenic method proposed in the present work to prepare highly visible
light active Ag@TiO2 NPs can be used in industrial effluent treatment as
well as in biomedical applications.
The anticancer activities of Ag@TiO2 and TiO2 have been evaluated
in vitro against A549 human lung carcinoma cell lines. After 24 h, cell
viability has been measured in the concentration range of 10–200 μg.
When the doping concentration of Ag@TiO2 was increased, the cell
viability was decreased. IC50 values of green synthesized Ag@TiO2 and
TiO2 were found to be 115 μg/mL (0.015 M), 138 μg/mL (0.010 M),
155 μg/mL (0.005 M) and 200 μg/mL, respectively as shown in sup-
plementary file (Fig. S4). The IC50 value was low only for 0.015 M
Ag@TiO2.
When doping concentration increased, green synthesized Ag@TiO2
exhibited good anticancer activities compared to TiO2. The excess
electron production was directly proportional to the enhanced pro-
duction of ROS (given in Fig. 6(a)), and it was used to break the A549
cell walls. Fig. S4 anticancer activity of (A) TiO2 (B) 0.005, 0.010 and
0.015 M Ag@TiO2 for A549 lung cancer cell lines.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jphotobiol.2019.111636.
3.11. Morphological Evidence of Apoptosis Analysis of Ag@TiO2 and TiO2
NPs
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