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Research Journal of Chemistry and Environment______________________________________Vol. 24 (4) April (2020)
Res. J. Chem. Environ.
129
Effect of Organic Doping TiO2 Nanoparticles on
Catalase and Peroxidase Activity and New Cancer
Treatment Approach
Asmaa J.AL-Lamei*, Zahraa K. Al-Hassani*, Muna Ali Shaker and Salmaa Abdul-Radah
Chemistry Department, College of Science, Al-Mustansiriyah University, IRAQ
Chemistry Department, Faculty of Pharmacy, Jabir ibn Hayyan Medical University, IRAQ
*asas.jameil@uomustansiriyah.edu.iq; Zahraa_kadhim@yahoo.com
Abstract
This study describes the synthesis of organic compound
doping TiO2 nanoparticles by sol-gel technique. By
spectral techniques, particle size was found around
73.34 nm at 90ᵒC.The nanoparticles are characterized
by XRD, SEM, AFM and FTIR. The inhibition effect of
doping nanoparticles on Catalase and Peroxidase is
also studied. Damage in DNA is responsible for cancer
formation and progression. This study used different
concentration of doping NPs.
The inhibition effect of doping nanoparticles on
catalase and enzymes is also studied by using different
concentration of doping NPs. It is found that the
activity of catalase and peroxidase enzymes increases
with decrease in nanoparticles concentrations and
decreases in inhibition percentage. Kinetic properties
of catalase and peroxidase activities are revealed by
doping NPs non-competitive type of inhibitors and
peroxidase activity competitive inhibitors. Titanium
nanoparticles showed a high significant percentage of
inhibition.
Keywords: Organic doping TiO2, sol-gel method,
catalase, peroxidase.
Introduction
Titanium dioxide or titania is a metal oxide semiconductor
with the tentative formula TiO2. Like other metal oxides, it
is hard, thermally stable and chemically resistant1,2.
Nanoscale titanium dioxide may be synthesized by number
of different methods including sol-gel, solvothermal
synthesis, direct oxidation, chemical or physical vapor
deposition, electrodeposition and microwave synthesis.3,4
The sol-gel method is the most popular method in current
literature.5
Doping is one of the most extensively-studied methods by
which TiO2 has been modified in the literature and its
practice is so wide-spread that its application to any existing
or newly-discovered photo catalysts has been described as
inevitable".6 Titanium dioxide materials may be doped using
a number of methods including ion implantation, magnetron
sputtering, sol-gel modification and annealing in gas7.
Doping may occur in one of two forms: either substitutional
in which the dopant atom replaces a lattice atom, or
interstitial, in which it is located between existing lattice
atoms.
The catalase (oxidoreductase ( is an enzyme that is
widespread in living organism )microorganisms, animals,
anaerobic organisms, plant tissue 8 and fungi).9 It exists in
big concentrations in the liver of mammals.10 Its functions
include stimulating hydrolysis of hydrogen peroxide into
oxygen and water.11 It is an important enzyme that protects
cells from oxidative damaging by reactive oxygen. Only one
molecule can convert millions of H2O2 to water and oxygen
per second.12
Catalase is a tetrameric protein with a quaternary structure
of four subunits of four polypeptide chains, each one
contains more than 500 amino acids.13 It contains four heme
(iron) groups that allow to react with the H2O2.14 It can
metabolize small molecules such as hydrogen peroxide,
ethyl hydroxyl peroxide and methyl hydroxyl peroxide, but
not bigger molecules such as lipid hydroxyl peroxide
produced from peroxidation of lipids.15
Peroxidase is widely spread in nature (in animal and plant
cells); it consists of three main categories (plant and animal
peroxidase and catalase).16 The optimal substrate is H2O2 but
others are more active with organic hydroxides such as lipid
peroxides. Peroxidase can contain an active cystine or
selenocystein residues.17 These enzymes are used with
hydrogen peroxide to stimulate the oxidation of organic and
inorganic compounds. The reactions of peroxidase can be
characterized by oxidative halogenation and
dehydrogenation, the transport of oxygen and break down of
hydrogen peroxide.18
Material and Methods
1) Synthesis of organic compound doping TiO2
Nanoparticle: Organic compound /TiO2 were prepared by
Sol-Gel technique19.
2) Determination of the activity of serum peroxidase by
using colorimetric method20: In a test tube 1.4 ml of phenol
solution was added with 1.5 ml of peroxide (substrate
0.017mmol/L), then the solution was incubated for about
three minutes, then 100 µl of serum + 20 µL distilled water
were added to the solution.
3) Determination the activity of serum catalase by using
manual method21: Catalase reacts with H2O2 (substrate),
residual H2O2 reacts with ammonium molybdate to get a
Research Journal of Chemistry and Environment______________________________________Vol. 24 (4) April (2020)
Res. J. Chem. Environ.
130
colored complex. From stock solution (100 µg/ml), different
concentrations of nano particles (20 ,30, 40,50) µg/ml were
prepared. The same steps were performed with replacement
of the volume of distilled water by nanoparticles to
determine the evaluation of the effect of nanoparticles on
enzyme activity. The inhibition percentages of both enzymes
were calculated according to the equation:
% inhibition = 100 - (the activity in the presence of
inhibitor/the activity in the absence of inhibitor) X 100
The activity of enzymes was determined with and without
the inhibitor, the values of Vmax maximal velocity and km
Michael constant were calculated by using the Lineweaver
– Burk equation.
Characterizations: The identification phase, particle size
and crystalline structure analysis are determined by XRD
using Shimadzu –6000 model with a Cu radiation ( λ= 1.54
Aᴼ),voltage 40 Kv and current 30 mA with speed5ᴼ /min.
The Atomic force microscopy(AFM)CAPM type AA3000 is
used to investigate the particle size and morphology of the
derived nanoparticles.
Results and Discussion
FTIR Spectral of TiO2/ organic compound (AQ)
nanoparticle: The FTIR spectra of nanoparticles of
anthraquinone and TiO2 were recorded to study the
functional group and binding sites. FTIR spectra of AQ are
show in figure 1. The assignments of absorption band in the
spectra at the band located at 1672.3 cm-1 are due to the
stretching vibration of the C= O bond. The band at 1631.83
cm-1 in spectra is due to the stretching vibration of the C=C
bond. It can be seen in the band at 1573.97 cm-1 indicating a
presence of C=C bond. The FTIR spectra of TiO2/AQ are
shown in figure 2. The band appearing at 633,603 cm-1
attributed to Ti-O bending mode. The band at 1450.22 cm-1
corresponds to the Ti-O-Ti bending while the bands at 1672
and 1573cm-1 have been reduced after nanoparticle TiO2/
AQ formation. The band at 1406.15 cm-1 very small.
AFM analysis: The AFM measurements can be considered
as the values obtained from SEM have some advantage of
SEM using the possibility to obtain statistical information
from any location in comparatively short time. Figure 3
showed that the average diameter of the particles was 73.34
nm; this diameter was larger than the value obtained from
SEM image analysis (65 nm). Root Mean square (RMS)
roughness of the nanoparticle could be obtained from AFM
analysis. RMS was found to be 2.33 nm on the plain TiO2/
AQ surface. Farhan et al22 synthesized α-Fe2O3 NPs by sol-
gel method using malic acid as a chelating agent and found
that the particles size of NPs was approximately 82nm.
Table 1
Different reagents used in titration
The reagents
Test
Blank No. 1
Blank No. 2
Blank No. 3
Serum
0.2ml
----
---
---
Substrate (H2O2)
1ml
1ml
1ml
---
D.W
20µl
20µl
20µl
20µl
Buffer (sodium phosphate) pH 7.4
----
---
---
1ml
Ammonium Molybdate
1ml
1ml
1ml
1ml
Buffer (sodium phosphate) pH 7.4
----
---
0.2ml
0.2ml
Serum
----
0.2ml
---
---
Figure 1: FTIR spectrum of AQ
Research Journal of Chemistry and Environment______________________________________Vol. 24 (4) April (2020)
Res. J. Chem. Environ.
131
Figure 2: FTIR spectrum of TiO2 /AQ nanoparticle
Figure 3: AFM topography map of TiO2/AQ nanoparticle
Research Journal of Chemistry and Environment______________________________________Vol. 24 (4) April (2020)
Res. J. Chem. Environ.
132
X-rays Diffraction of TiO2/ organic compound: As it is
indicated in figure 4, the diffraction peaks can be indexed as
the anatase phase for TiO2 according to the JCPDS card no.
21-1276.The main diffraction peaks at 2 θ = 25.34,
37.80,53.80,48.07 and 55.07 0 belong to(101),(004), (105),
(200) and (211) planes of the TiO2 tetragonal phase. Figure
4 shows a strong peak occurring at 2 θ = 25.34O which
corresponds to the (101) reflection, while other characteristic
peaks (004), (105), (200) and (211) correspond to different
crystalline planes.
In case of the sample obtained with the use of the AQ, the
XRD patterns show a weak peak occurring at 2 θ =23.5, 27.4,
62.7, 68.9, 70 and 75.120 corresponding to the AQ structure.
Analysis of the data leads to the conclusion that the addition
of AQ at the stage of titanium dioxide synthesis leads to a
mixed anatase and AQ structure caused significant changes
in crystallites characteristic. It means samples prepared with
the smallest addition of AQ are characterized with the
biggest crystallite sizes calculated using Scherrer formula
(24.19 nm).
Scanning Electron Microscopic (SEM): The SEM
morphologies of the nano crystalline AQ doped TiO2 sample
is depicted in figure 5 showing SEM image of the prepared
nanoparticle obtained by sol- gel method the synthesized
anthraquinone doped TiO2 showing spherical structure like
morphology .The average diameter of TiO2 /AQ
nanoparticle is clear.
Kinetic and activity of enzymes: The effect of nanoTiO2
doping of serum catalase(CAT) and peroxidase(POD)
enzymes activity was investigated in this study. The
biochemical tests revealed that nanoparticles caused
inhibitory effects on catalase and peroxidase enzymes. The
relationship between doping nanoparticles concentration
versus the activity of enzymes was shown in table 2 and table
3 for catalase and peroxidase respectively. These results
observed that any increase in nano concentrations caused
decrease in activation of enzymes and increase in inhibition
percentage, the activity of catalase was affected and
decreased from 42.81 IU/ml to 16.41 IU/ml and for
peroxidase decreased from 154.3 IU/ml to 44.9 IU/ml at 50
µg\mL.
The result showed that titanium nanoparticle exhibited a
significantly higher percentage of inhibition and it increased
with increasing the concentration. Under the same
conditions and at different concentrations of titanium
nanoparticle, determine inhibition of type (by using
Lineweaver-Burk equation) and evaluate the values Vmax,
Km and the type of inhibition. The results have revealed that
catalase has the type of inhibition as shown in table 3 and
figure 6 as uncompetitive inhibition with titanium doping
nanoparticle, with Vmax 33.33(mol/ml/min) and Km 50, but
peroxidase has competitive inhibition with titanium doping
nanoparticle with Vmax 250 (mol/ml/min) and Km 0.0128
as shown in figure 7.
In this study, the results showed that titanium nanoparticles
have a high effect on both enzymes and it supports the use
of titanium nanoparticles that inhibited both of enzymes and
exhibited significant free radical scavenging and antioxidant
activities which can be related to treatment of inflammation.
For cancer, nanotechnology offers great opportunities for
delivery of specific drugs to disease sites, so it has many
applications in the medical field, because nanoparticles have
their potential impact in cancer treatment which gives a
promising future for nanotechnology in medicine.23
Nanoparticles have a unique approach to cancer treatment.
A large number of nanoparticle delivery systems have been
developed for the treatment of cancer.24 Some nanoparticles
show anti-inflammatory effects25 and suppression of
infection26. We assumed that titanium nanoparticles interact
with the functional groups of catalase and peroxidase to get
denaturing and disruption of protein and they cause the
inhibitions of enzymes. We recommend using titanium
nanoparticles to treat some disease such as inflammation and
cancer because it has an effect on inhibition of enzymes
catalase and peroxidase.
Figure 4: X-ray diffraction (XRD) of TiO2 /anthraquinone nanoparticle
Research Journal of Chemistry and Environment______________________________________Vol. 24 (4) April (2020)
Res. J. Chem. Environ.
133
Figure 5: A, B SEM image of TiO2 /AQ nanoparticle
Table 2
The effect of different concentrations of Titanium doping nanoparticles on the activity of Catalase in human serum.
Nanoparticles con. (µg/ml)
Catalase activity IU/ml
Inhibition %
Nil
42.81
0.00
20
35.16
17.86
30
32.11
24.99
40
21.03
50.87
50
16.41
61.66
Table 3
The effect of different concentrations of Titanium doping nanoparticles on the activity of Peroxidase in human serum.
Nanoparticles con. (µg/ml)
Peroxidase activity IU/ml
Inhibition %
Nil
154.3
0.00
20
133.7
13.35
30
82.5
46.53
40
76.2
50.61
50
44.9
70.9
Table 4
The kinetic parameter Km, Vmax and type of inhibition for Titanium doping nanoparticles with
catalase and peroxidase
Figure 6 : The Lineweaver – Burk plot in the presence and abcence of titanium doping nanoparticles with catalase
Enzyme
Km (M)
Vmax
(mol/ml/min)
Type of
Inhibition
Catalase
50
33.33
Uncompetitive
Peroxidase
0.0128
250
Competitive
200 nm
A
200 nm
B
Research Journal of Chemistry and Environment______________________________________Vol. 24 (4) April (2020)
Res. J. Chem. Environ.
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Figure 7: The Lineweaver – Burk plot in the presence and abcence of Titanium doping nanoparticles
with peroxidase
Conclusion
In the present work the TiO2/organic compound
nanoparticles were synthesized by sol-gel method at 90ᵒC.
In AFM analysis, the particle size of produced TiO2/organic
compound was approximately 73.34 nm. From the FTIR
spectrum of TiO2/doping NPs, the peak at 633.603cm-1 was
attributed to Ti-O bending mode. The band at1450.22cm-1
corresponds to the Ti-O-Ti bending while the bands at 1672
and 1573cm-1 have been reduced after nanoparticle TiO2
doping formation. The band at 1406.15 cm-1 is very small.
This study evaluated the effect of
nanoparticalesTiO2/organic compound and found the
inhibitory effects on catalase and peroxidase enzymes.
The greater inhibition of nano was demonstrated at
concentration 50µg/ml. Inhibitor percentage of catalase
enzyme exhibits 61.66% lower than peroxidase enzyme
70.9%. By increasing the concentration of titanium
nanoparticles, the inhibition effect was enhanced. We can
say that according to the results, when titanium nanoparticles
are used for cancer treatment, they will cause elevation of
free radical levels, therefore we recommend giving
antioxidant agents in companion with these nanoparticles.
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1\v
1\[s]
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Research Journal of Chemistry and Environment______________________________________Vol. 24 (4) April (2020)
Res. J. Chem. Environ.
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(Received 08th June 2019, accepted 10th August 2019)
