Materials Science and Engineering C 62 (2016) 919–926

Contents lists available at ScienceDirect

Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

ZnO nanopellets have selective anticancer activity

Prashanth Gopala Krishna a,b, Prashanth Paduvarahalli Ananthaswamy b,c,⁎, Tejabhiram Yadavalli d,
Nagabhushana Bhangi Mutta e, Ananda Sannaiah f, Yogisha Shivanna g
a
Department of Chemistry, Sir M. Visvesvaraya Institute of Technology, Bengaluru 562157, India
b
Research and Development Centre, Bharathiar University, Coimbatore 641046, India
c
Department of Chemistry, Sai Vidya Institute of Technology, Bengaluru 560064, India
d
Nanotechnology Research Centre, SRM University, Chennai 603203, India
e
Department of Chemistry, M. S. Ramaiah Institute of Technology, Bengaluru 560054, India
f
Department of Chemistry, University of Mysore, Mysuru 560006, India
g
Drug Discovery Research and Development Centre, Skanda Life Sciences Private Limited, Bengaluru 560091, India

a r t i c l e i n f o a b s t r a c t

Article history: This research work presents the synthesis of ZnO nanopellets (ZNPs) by low temperature hydrothermal
Received 16 November 2015 approach and evaluation of their antibacterial activity, cytotoxicity in vitro and in vivo. Structural and morpholog-
Received in revised form 10 January 2016 ical studies conducted on the sample reveal hexagonal ZNPs in the size range of 250–500 nm. Surface area
Accepted 13 February 2016
measurements showed high porosity of the sample compared to conventional ZnO nanoparticles. Antimicrobial
Available online 15 February 2016
studies revealed their bactericidal nature against both Gram-negative and Gram-positive bacteria. Furthermore,
Keywords:
to better understand the parameters that affect the interactions between our ZNPs and mammalian cells, and
ZnO nanopellets thus their biocompatibility, we have examined the impact of cell culture conditions as well as of material
Antibacterial properties on cytotoxicity by DPPH, blood hemolysis and MTT assay. The results showed good antioxidant
DPPH assay capacity and biocompatibility of ZNPs at higher concentrations. MTT assay revealed the anticancer activity of
MTT assay ZNPs against prostate and breast cancer cell lines. Acute toxicity tests on Swiss albino mice showed no evident
Hemocompatibility toxicity over a 14 days period.
In vivo acute toxicity © 2016 Elsevier B.V. All rights reserved.

1. Introduction been developed to synthesize ZnO nanoparticles such as precipitation,

ZnO is an inorganic antimicrobial agent which is generally recognized
as safe (GRAS) under US-FDA listings, to human beings and animals. ZnO
nanoparticles have been used in the field of textiles, catalysis, solar cells,
food packaging materials, cosmetics, antimicrobials, leather industries,
etc. [1–3]. ZnO nanoparticles have also received significant attention in
the field of cancer therapy in the recent past [4]. ZnO nanoparticles of
varying shapes such as spherical, polygonal and rod have been prepared
with different particle sizes ranging from 5–175 nm and their cytotoxicity
has been studied on murine and cancer cells such as HepG-2, MCF-7,
Colo320, HeLa, cloudman S91, tumor U87 [5–8]. Studies have shown
that ZnO nanoparticles cause cytotoxicity to many types of cells such as
human kidney cells, human hepatocytes, embryonic kidney cells, HT29
and Caco-2 cell lines of epithelial origin, glioma cells [9–12]. Recently,
size controlled synthesis of ZnO nanomaterials of desired size and shapes
have been the subject of investigation by researchers because it has
been the subject to intrinsic scrutiny, given the numerous properties
that could be controlled by size and shape. Several methods have
⁎ Corresponding author at: Department of Chemistry, Sai Vidya Institute of Technology,
Bengaluru 560064, India.
E-mail address: prsnthmysore@gmail.com (P. Paduvarahalli Ananthaswamy).
hydrothermal, combustion, sol–gel, chemical vapor deposition, spray
pyrolysis, sonochemical, etc. [13–16].
Of the various methods, hydrothermal synthesis has been considered
to be the most dependable for the production of nanoparticles with con-
sistent size because of its ability to control the particle size by controlling
the synthesis conditions such as temperature, pressure, etc. [17–19].
Despite the widespread applications of ZnO nanoparticles, the safety
of this metal oxide is still under scrutiny. Although many articles have
been reported on the acute toxicity of zinc oxide nanoprticles, the
same for zinc oxide nanopellets in the size range of 250–500 nm has
not been discussed so far. As the interaction of nanoparticles with
microorganisms and biomolecules is an expanding area of research
which is still largely unexplored, this study reports the synthesis of pel-
let shaped ZnO nanoparticles by simple, low temperature hydrothermal
method and their characterization by different techniques. The synthe-
sized ZNPs were evaluated for their antimicrobial efficacy against both
Gram-negative and Gram-positive bacteria Pseudomonas aeruginosa,
Escherichia coli, Staphylococcus aureus and Bacillus subtilis by well diffu-
sion technique, antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl
hydrate (DPPH) assay, in vitro cytotoxicity by blood hemolysis, in vitro
anticancer activity in prostate cancer cell line PC-3, colorectal cancer
cell line HCT116, lung cancer cell line A549 and breast cancer cell line

http://dx.doi.org/10.1016/j.msec.2016.02.039
0928-4931/© 2016 Elsevier B.V. All rights reserved.
920 P. Gopala Krishna et al. / Materials Science and Engineering C 62 (2016) 919–926

Table 1 fourier transform infrared spectroscopy (FTIR) which was carried out
Grouping of animals for the assessment of acute toxicity of ZNPs. on Perkin–Elmer spectrophotometer (Model: Spectrum 1000) using
Group No. Animals Animal numbers Treatment Dose KBr disk method within the range of 350–3000 cm−1. Surface morphol-
(mg/Kg, body weight) ogy of the sample was studied by field emission scanning electron mi-
IA SA mice A1 to A3 Test group 5 croscopy (FE-SEM) performed on FEI Quanta FEG 200 — high
IB SA mice B1 to B3 Test group 5 resolution scanning electron microscope equipped with energy disper-
IIA SA mice A1 to A3 Test group 50 sive x-ray spectrometer (EDS). The shapes and particle size were in-
IIB SA mice B1 to B3 Test group 50
vestigated by high resolution transmission electron microscopy
IIIA SA mice A1 to A3 Test group 300
IIIA SA mice B1 to B3 Test group 300 (HRTEM) carried out on JEOL 3010 instrument with a UHR pole
IVA SA mice A1 to A3 Test group 2000 piece. Brunauer–Emmett-Teller (BET) surface area measurements
IVB SA mice B1 to B3 Test group 2000 were carried out on Micromeritics ASAP 2020.

2.3. Evaluation of antibacterial activity

MDA-MB-231 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. ZNPs were tested on Swiss albino mice (SA Mice)
for their in vivo acute toxicity over a period of 14 days.
2. Materials and methods
2.1. Synthesis of ZNPs
All the chemicals and reagents used in this study were of analytical
grade without further purification. 7 g of zinc nitrate hexa hydrate
[Zn(NO3)2.6H2O] was dissolved in 40 mL of double distilled water.
1.0 N NaOH was added till pH of the solution reached 12. The solution
was stirred for 15 min, transferred in to a 100 mL Teflon-lined stainless
steel autoclave, sealed and was maintained at an external temperature
of 180 °C for 16 h and then allowed to cool naturally to room tempera-
ture. The resulting precipitate was washed several times with double
distilled water and then with absolute ethanol, to remove the ions
adhering to the final product. The product was dried at 110 °C in hot
air oven for 2 h and further cooled to room temperature.
2.2. Characterization techniques
The crystalline phase and crystal structures of ZNPs were studied by
X-ray diffraction using Panalytical X'pert diffractometer with Cu Kα ra-
diation (λ = 1.5418 Ǻ) as the source at a scanning speed of 0.05° 2-θ
step size and 2 s per step. The formation of ZnO and the absence of
any other functional groups from the precursors were confirmed using
The antibacterial activity of ZNPs was carried out by well diffusion
method in Mueller Hinton (MH) agar media against Gram-negative
P. aeruginosa, E. coli and Gram-positive S. aureus, B. subtilis bacterial
cultures. The bacteria were cultured overnight at 37 °C in MH and
adjusted to a final density of 107 CFU/mL by 0.5 McFarland standards.
About 25 mL of molten MH agar was poured into sterile petri plates.
The plates were allowed to solidify, after which 100 μL of the pathogenic
bacteria cultures were transferred onto plate and made culture lawn by
using sterile L-rod spreader. Homogeneous dispersions of ZNPs with
different concentrations ranging from 1 mg/mL to 62.5 μg/mL (with
two fold dilution) were prepared by ultrasonication. Wells were cut
and dispersions of ZNPs (of different concentrations) were loaded. The
plates were incubated at 37 °C for 24 h. The antibacterial activity was
determined by measuring the diameter of the zone of inhibition formed
around the wells.
2.4. Assessment of antioxidant activity by DPPH assay
DPPH radical has a deep violet color in solution, and gradually it
becomes colorless or pale yellow in the presence of ZNPs. This property
allows visual monitoring of the reaction and the concentration of radi-
cals is monitored from the change in percentage of absorption [20].
The antioxidant activity of ZNPs was measured by standard DPPH meth-
od [21,22]. In brief, 3.94 mg of DPPH was dissolved in 10 mL of methanol
to obtain1 mM DPPH solution. 860 μL of 50% methanol dispersed with
ZNPs (with different concentrations of 1.5, 3.0, 6.0, 12.0, 24.0 mg/mL)

Fig. 1. Flow chart for acute oral toxicity study as per OECD 423 guidelines.
 P. Gopala Krishna et al. / Materials Science and Engineering C 62 (2016) 919–926 921

Table 2
Results of antibacterial activity of ZNPs (Zone of inhibition in mm).

Concentration of ZnO suspensions (/mL)

Pathogen 1 mg 500 μg 250 μg 125 μg 62.5 μg Positive control (Ciprofloxacin-100 μg/mL)

P. aeruginosa 14.00 ± 0.025 12.00 ± 0.000 10.00 + 0.000 0.00 + 0.000 0.00 + 0.000 40.00 + 0.500
E. coli 19.50 + 1.250 16.00 + 0.250 15.00 + 0.825 14.00 + 0.000 12.00 + 1.000 35.25 + 0.125
S. aureus 29.00 + 0.125 27.25 + 0.000 24.50 + 0.125 21.00 + 0.000 18.75 + 0.125 35.00 + 0.125
B. subtilis 32.00 + 0.00 28.25 + 0.000 22.00 + 0.000 20.00 + 0.125 18.00 + 0.000 30.50 + 1.000

was mixed with 140 μL of 1 mM DPPH solution and incubated at 37 °C
for 30 min. The absorbance was recorded at 520 nm against 50% meth-
anol blank. A control reaction was carried out without the test sample.
Gallic acid [3,4,5-trihydroxy benzoic acid] was used as the reference
standard.
2.5. In vitro cytotoxicity by hemolysis testing
The hemolysis activity test was performed against ZNPs by following
the procedure of Dhaneswar Das et al. [20]. In brief, to 9 mL of the blood
sample collected from a sheep, 1 mL of 3.8% sodium citrate was mixed.
This inhibits the coagulation of blood. The sample was centrifuged at
3000 rpm for 5 min. The supernatant which was having platelet poor
plasma was discarded. The pellet containing RBC was suspended in
10 mL of phosphate buffer saline (PBS) of pH 7.4. The cells were
suspended in PBS to obtain a uniform suspension of cells. ZNPs with
different concentrations of 0.25, 0.5, 1.0, 2.5, 5.0, 10.0 mg/mL were
taken in different test tubes. To all the test tubes 2 mL of erythrocyte
suspension was added and the test tubes were inverted. The tubes
were then gently shaken to retain the contact of the blood with ZNPs
and incubated at 37 °C for 90 min. The samples were centrifuged at
3000 rpm for 5 min to pellet out the RBC cells. The supernatant was
then separated and the absorbance was measured at 540 nm using a
UV–Vis spectrophotometer against a PBS blank solution.
The percentage of hemolytic index (%) was calculated by using the
following formula [23].

orally to a group of experimental animals at one of the defined doses.
ODtest sample – ODnegative control
Hemolysis ð%Þ ¼
 100
ODpositive control – ODnegative control
Where, OD is the optical density value. Triton X-100 and PBS served
as positive and negative controls respectively.
2.6. Anticancer activity by MTT assay
Anticancer activity of ZNPs was carried out by MTT assay as reported
in the literature with some modifications [24–30]. Cell lines HCT116,
PC-3, A549 and MDA-MB-231 (procured from ATCC) of 80% confluent
were trypsinized. The viable 20,000 cells/well were seeded in a 96
well plate and incubated for 24 h at 37 °C, 5% CO2 incubator. ZNPs
from 0–300 μg/mL in Dulbecco's Modified Eagle's Medium without
fetal brovine serum were incubated for 24 h. After incubation with
ZNPs the media was removed from the wells and 100 μL/well of the
MTT (5 mg/10 mL of MTT in 1× Phosphate buffered saline, the solution
was filtered through 0.2 μM filter) working solution was added and
Table 3
Hemolysis of blood by ZNPs.
Concentration Percent hemolysis
of ZNPs (mg/mL)
0.25 0.22
0.5 0.60
1.0 1.71
2.5 2.83
5.0 3.95
10.0 5.16
incubated for 3 to 4 h. After incubation with MTT reagent, the media
was removed from the wells and 100 μL of dimethyl sulfoxide was
added to rapidly solubilize formazan and absorbance was measured at
590 nm. Percent of inhibition was calculated as [100 − (As/
(Ac) × 100] and cell viability was calculated as [As × 100/Ac] where, As
and Ac are the absorbance values of the sample and control respectively.
2.7. Assessment of in vivo acute toxicity
Acute toxicity studies were carried out as per the organization
for economic co-operation and development (OECD) 423 protocols.
The animals used were SA mice of weight 25 ± 5 g. The animals were
of both the sex. Their age at the start of the studies was 8–10 weeks.
3 animals/dose levels were studied. The acclimatization was one week
prior to dosing. The animals were identified by cage numbers and
markings on animals. The route of administration was per os (by
mouth). The animals were housed in separate cages under controlled
conditions of temperature 22 ± 2 °C. All animals were given standard
diet and water regularly.
Table 1 shows the grouping of animals for the acute oral toxicity
studies of ZNPs. Methodology followed was as per the guidelines of
OECD 423. In brief, the acute toxic class method set out in this guideline
is a stepwise procedure with the use of 3 animals of a single sex per step.
Depending on the mortality and or the moribund status of the animals,
on average 2–4 steps may be necessary to allow judgment on the acute
toxicity of the test substance. The substance, ZNPs were administered
The substance was tested using a stepwise procedure, each step using
three animals of a single sex (normally female). Absence or presence
of compound-related mortality of the animals dosed at one step will
determine the next step, i.e.; no further testing is needed, dosing of
three additional animals, with the same dose and, dosing of three
additional animals at the next higher or the next lower dose level.
Three animals were used for each step. The dose level to be used as
the starting dose was selected from one of the four fixed levels, 5, 50,
300 and 2000 mg/kg body weight. Flow chart for acute oral toxicity
study as per OECD 423 guidelines is as shown in Fig. 1.
3. Results and discussion
3.1. Crystal structure
The PXRD pattern of ZNPs is presented in Fig. 2. The diffraction
pattern agrees with the standard Joint Committee on Powder
Table 4
Observations of in vivo acute toxicity studies of ZNPs.
Group No. Dose (mg/Kg) Lethality
IA 5 0/3
IB 5 0/3
IIA 50 0/3
IIB 50 0/3
IIIA 300 0/3
IIIB 300 0/3
IVA 2000 0/3
IVB 2000 0/3
922 P. Gopala Krishna et al. / Materials Science and Engineering C 62 (2016) 919–926

3.3. Product formation mechanism

A probable reaction mechanism is proposed for the formation of ZnO

nanoparticles by hydrothermal method. Studies have shown that the
anisotropic growth of ZnO is favored when the pH of the medium is
higher than 7.0 [32]. Thus our reaction condition (pH) may favor aniso-
tropic non-uniform growth of the nanoparticles. According to the liter-
ature, in this reaction source of Zn is in the forms of Zn(OH)2 and [Zn
(OH)4]2−. Zn(OH)2 under the hydrothermal conditions dissolves to a
considerable extent to form Zn2 + and OH− ions, once the product of
[Zn2+] and [OH−] exceeds a critical value which is necessary for the for-
mation of ZnO crystals, the ZnO crystals precipitate from the solution. As
the solubility of ZnO is very less than that of Zn(OH)2 under the
hydrothermal conditions, Zn(OH)2 strongly tends to be converted in
to ZnO crystals during the hydrothermal process [33–36].
Probable reaction mechanism of formation of ZnO is as follows.

Zn(NO3)2·6H2O + 2NaOH → Zn(OH)2 + 2NaNO3 + 6H2O

Fig. 2. PXRD pattern of ZNPs.

Zn(OH)2 + 2H2O → [Zn(OH)4]2− + 2H+

Diffraction Standards (JCPDS) No. [36–1451] corresponding to

zincite pattern and can be indexed as hexagonal wurtzite type of [Zn(OH)4]2− → ZnO + H2O + 2OH−
ZnO. Absence of other impurity peaks indicates high purity of the
ZNPs. The average crystallite size D was calculated using the Scherrer
or
equation, D = k λ/β Cosθ, where k is the Scherrer constant, λ is the
wavelength of the X-ray used (1.54 Ǻ), θ is the Bragg angle and β is
Zn(OH)2 → Zn2+ + 2OH−
the full-width at half-maxima (FWHM) of the diffraction peaks and
it was found to be ~ 31 nm.

Zn2+ + 2OH− → ZnO + H2O

3.2. FTIR analysis

The FTIR spectrum of ZNPs is shown in Fig. 3. The transmittance 3.4. Surface area measurements
band at 445 cm−1 corresponds to the Zn–O bonding and confirms the
presence of ZnO particles. The FTIR result confirms the high purity of The surface area of ZNPs was measured by the standard BET
ZNPs. Small absorption bands near 1114, 1398, were observed. These technique with N2 adsorption–desorption isotherms on Micromeritics
were likely due to CO2 (C = O) and H2O (O–H) absorbed from the atmo- ASAP 2020. The BET surface area value of ZNPs was found to be
sphere and therefore can be neglected. A weak band observed at around 22.79 m2/g.
2340 cm−1 also might be attributed to the vibrations of atmospheric
CO2 [31]. 3.5. Morphological studies

The surface morphology of ZNPs was studied using the scanning

electron microscopy. The FE-SEM image of ZNPs shown in Fig. 4
(A) reveals that the particles have non-uniform pellet like morphology.
Fig. 4 (B) shows the HRTEM image of ZNPs. It confirms that the particles
are pellets and rod shaped with the particle size in the range of
250–500 nm. EDS analysis for the nanopowder shown in Fig. 5
indicates the dominant composition of ZnO.

3.6. Antibacterial assay

The effect of ZNPs on different organisms is shown in Fig. 6 and the

results are presented in Table 2. These results show that the zone of
inhibition is maximum for ZNPs against Gram-positive bacterial
cultures than the Gram-negative bacteria used in our studies. This result
might be indicative of higher Gram-negative strain tolerance against
ZNPs over Gram-positive bacterial strains. These results are in agree-
ment with the literature which reports that the ZnO nanoparticle effect
is more pronounced against Gram-positive bacterial strains than Gram-
negative bacterial strains [13,31]. Furthermore, these results also sug-
gest that ZNPs are not only toxic to Gram-positive bacteria, but also to
Gram-negative bacteria.
The detailed mechanism of the bioactivity of ZnO is still under
Fig. 3. FTIR spectrum of ZNPs. discussion. Many mechanisms have been proposed related to this. The
 P. Gopala Krishna et al. / Materials Science and Engineering C 62 (2016) 919–926 923

Fig. 4. (A) FE-SEM and (B) HR-TEM images of ZNPs.

main two probable mechanisms involved in the interaction between
nanoparticles and bacteria suggested by several investigations are
(1) the production of increased levels of reactive oxygen species, mostly
hydroxyl radicals and singlet oxygen [37,38–40] and (2) Zinc toxicity of
cell membrane by adhesion of ZnO particles which inhibits the bacterial
growth [41,42].
3.7. DPPH free radical scavenging assay
for DPPH radical scavenging activity of ZNPs and standard (gallic acid)
were derived from a nonlinear regression analysis (curvefit) based
on sigmoidal dose response curve (variable) and computed using
GraphPad Prism 5. The IC50 values for ZNPs and gallic acid were found
to be 11,408 μg/mL and 1.667 μg/mL respectively. The antioxidant activ-
ity of ZNPs might be due to the transfer of electron density located at ox-
ygen to the odd electron located at nitrogen atom in DPPH resulting the
decrease in intensity of n → π⁎ transition [20].

The color of DPPH solution gradually changes from deep violet to
pale yellow in the presence of ZnO nanoparticles. The percentage of
DPPH scavenging was estimated from the decrease of absorption at
520 nm, which represents the amount of DPPH in the solution. DPPH
free radical scavenging activity of ZNPs and gallic acid with their differ-
ent concentrations is presented in Fig. 7. From these studies, IC50 values
3.8. Cytotoxicity by hemolysis
The effect of ZNPs on RBC is shown in Fig. 8 and the results of hemo-
lysis assay are presented in Table 3. Since 5% hemolysis is considered as
permissible limit for biomaterials, within the limitations of this study,
up to a concentration of 5.0 mg/mL of ZNPs can be taken for hemolysis

Fig. 5. EDS for ZNPs.

924 P. Gopala Krishna et al. / Materials Science and Engineering C 62 (2016) 919–926
Fig. 6. Zone of inhibition produced by (A–D) ZNPs against P. aeruginosa, E. coli, S. aureus, B. Subtilis respectively, (E–H) standard antibiotic against P. aeruginosa, E. coli, S. aureus, B. subtilis
respectively.
activity. Therefore, it can be concluded that ZNPs synthesized in our
present studies are biocompatible in nature up to a concentration of
5 mg/mL.
3.9. Anticancer activity
Cytotoxic effect of ZNPs in PC-3, HCT116, A549 and MDA-MB-231
cell lines is shown in Fig. 9 (A). In our studies it is evident that
HCT116, PC-3, A549 and MDA-MB-231 cells respond differently
to ZNPs exposure. Among the four cell lines selected for the study,
ZNPs induced highest toxicity in PC-3 and lowest toxicity in
HCT-116. The order of cytotoxicity effect of ZNPs was found to be
PC-3 N MDA-MB-231 N A-549 N HCT-116. This implies that type of
cell under investigation is important when the anticancer activity
of ZnO nanoparticles is considered.
IC50 value, the concentration of ZNPs needed to inhibit cell growth
by 50% for cytotoxicity test on PC-3 and MDA-MB-231 were derived
from nonlinear regression analysis (curve fit) based on sigmoid dose
response curve (variable) and computed using Graph Pad Prism 5. It is
shown in Fig. 9 (B) and (C) respectively. The IC50 value for PC-3 and
MDA-MB-231 were found to be 85.29 μg/mL and 98.22 μg/mL
respectively.
Literature shows that ZnO nanoparticles induce cytotoxicity in a cell
specific and proliferation dependent manner by rapidly dividing cancer
Fig. 7. DPPH free radical scavenging assay of ZNPs and gallic acid with their different.
concentrations.
cells being the most susceptible and quiescent cells being the least
sensitive [13,43]. However, the anticancer activity of ZnO nanoparticles,
in particular the mechanism of apoptosis in cancer cells due to
ZnO nanoparticles is still not clear. The results of ZNPs on PC-3 and
MDA-MB-231 are promising and hence the internal mechanism needs
to be discovered in the future.
3.10. Acute toxicity
The acute toxicity study was performed for 14 days. General behav-
ior and lethality were evaluated to assess the acute toxicity of ZNPs.
Observations of acute toxicity studies are presented in Table 4. As visu-
alized from it, no mortality and no toxicity were observed throughout
the dosing schedule of 14 days in all dose level in all groups. Thus
LD50 (lethal dose, 50%) cut off was calculated as 2000 mg/kg body
weight, as per the methodology adopted.
4. Conclusions
The present study was conducted for the evaluation of anticancer
activity and acute toxicity of ZNPs which were synthesized by hydro-
thermal method. Antibacterial studies indicate that ZNPs were not
only toxic to Gram-positive but also to Gram-negative bacteria.
Although this phenomenon has been observed in other reports, it is in-
deed of significance for nanopellets in the size range of 250–500 nm.
Antioxidant assay and blood hemolysis studies confirmed the bio
Fig. 8. Blood hemolysis by ZNPs.
 P. Gopala Krishna et al. / Materials Science and Engineering C 62 (2016) 919–926
Fig. 9. (A) Cytotoxic effect of ZNPs in PC-3, HCT116, A549 and MDA-MB-231 cell lines. Cells were treated with various concentrations (0, 2.4, 4.7, 9.4, 18.7, 37.5, 75, 150, 300 μg/mL)
of ZNPs for 24 h grown in a serum free media. The percentage of cell death induced was determined using the MTT assay, (B–C) Determination of IC50 values of ZNPs in PC-3 and
MDA-MB-231 respectively.
inhibitory ability and bio-compatibility of the sample respectively at
varied concentrations. Among PC-3, HCT116, A549 and MDA-MB-231
cell lines selected in our studies ZNPs exhibited highest toxicity in PC-
3 and lowest toxicity in HCT-116 based on the tumorigenic ability of
the cell lines. Further experimentation on the activity of these materials
shall be conducted on primary cell lines and co-cultures in order to
understand the efficacy of ZNPs on cancer cell lines. The acute toxicity
studies conducted in SA mice as per OECD 423 guidelines showed
no mortality and no toxicity throughout the dosing schedule of
14 days in all dose level in all groups. The LD50 cut off was calculated
as 2000 mg/kg body weight, as per the methodology adopted. In con-
clusion, we propose that the ZNPs developed in this report carry a
significant step towards the production of cheap, antimicrobial,
biocompatible and thoroughly effective anti-cancer agent..
Acknowledgments
The author G.K. Prashanth thanks the management of Sri KET
and Dr. M.S. Indira, Principal of Sir MVIT, Bengaluru for the support
and encouragement extended towards this project. The authors
thank Dr. Avinash Mahajan, Professor, IITB for PXRD and Dr. Vivek
Polshettiwar, TIFR, Mumbai for BET measurements. The authors
acknowledge Pinnacle Biomedical Research Institute, Bhopal for the
facility extended towards the animal studies [the animal experiment
925
was approved by institutional animal ethics committee (IAEC) of PBRI,
Bhopal (Reg. No. 1283/PO/c/09/CPCSEA), with the protocol reference
No. PBRI/IAEC/13-14/PN-395], Nanotechnology Research Center, SRM
University for FE-SEM and EDS analysis, IITM for HRTEM analysis.
References
[1] M.D. Newman, Mira Stottland, J.I. Ellis, The safety of nanosized particles in titanium
dioxide and zinc oxide based sunscreens, J. Am. Acad. Dermatol. 61 (2009) 685–692.
[2] Petya Petkova, Antonio Francesko, Margarida M. Fernandes, Enest Mendoza, Ilana
Perelshtein, Aharon Gedanken, Tzanko Tzanov, Sonochemical coating of textiles
with hybrid ZnO/chitosan antimicrobial nanoparticles, Appl. Mater. Interfaces 6
(2014) 1164–1172.
[3] H.R. Nawaz, B.A. Solangi, B. Zehra, U. Nadeem, Preparation of nano zinc oxide and its
application in leather as a retanning and antibacterial agent, Can. J. Sci. Ind. Res. 2
(2011) 164–170.
[4] Mohd Javed Akhtar, Maqusood Ahamed, Sudhir Kumar, M.A. Majeed Khan, Javed
Ahmad, S.A. Alrokayan, Zinc oxide nanoparticles selectively induce apoptosis in
human cancer cells through reactive oxygen species, Int. J. Nanomedicine 7
(2012) 845–857.
[5] L. Palanikumar, S. Ramasamy, C. Balachandran, Antibacterial and cytotoxic response
of nano zinc oxide in gram negative bacteria and colo 320 human adenocarcinoma
cancer cells, Curr. Nanosci. 9 (2013) 469–478.
[6] F. Namvar, H.S. Rahman, R. Mohamad, S. Azizi, P.M. Tahir, M.S. Chartrand, S.K. Yeap,
Cytotoxic effects of biosynthesized zinc oxide nanoparticles on murine cell lines,
Evid. Based Complement. Alternat. Med. 593014 (2015).
[7] R. Wahab, M.A. Siddiqui, Q. Saquib, S. Dwivedi, J. Ahmad, J. Musarrat, A.A. Al-
Khedhairy, H.-S. Shin, ZnO nanoparticles induced oxidative stress and apoptosis in
HepG2 and MCF-7 cancer cells and their antibacterial activity, Colloids Surf. B:
Biointerfaces 117 (2014) 267–276.
926 P. Gopala Krishna et al. / Materials Science and Engineering C 62 (2016) 919–926
[8] R. Wahab, N.K. Kaushik, A.K. Verma, A. Mishra, I.H. Hwang, Y.-B. Yang, H.-S. Shin,
Y.–.S. Kim, Fabrication and growth mechanism of ZnO nanostructures and their cy-
totoxic effect on human brain tumor U87, cervical cancer HeLa, and normal HEK
cells, J. Biol. Inorg. Chem. 16 (2011) 431–442.
[9] I. Pujalte, I. Passagne, B. Brouillaud, M. Treguer, E. Durand, C. Ohayon-Courtes,
Beatrice L'Azou, Cytotoxicity and oxidative stress induced by different metallic
nanoparticles on human kidney cells, Part. Fibre Toxicol. (2011) 8–10.
[10] R. Guan, T. Kang, F. Lu, Z. Zhang, H. Shen, M. Liu, Cytotoxicity, oxidative stress,
and genotoxicity in human hepatocyte and embryonic kidney cells exposed to
ZnO nanoparticles, Nanoscale Res. Lett. 7 (2012) 602.
[11] Tianshu Kang, Rongfa Guan, Xiaoqiang Chen, Yijuan Song, Han Jiang, Jin Zhao,
In vitro toxicity of different-sized ZnO nanoparticles in Caco-2 cells, Nanoscale
Res. Lett. 8 (2013) 496.
[12] J.W. Rasmussen, E. Martinez, P. Louka, D.G. Winget, Zinc oxide nanoparticles for se-
lective destruction of tumor cells and potential for drug delivery applications, Expert
Opin. Drug Deliv. 7 (2010) 1063–1077.
[13] M. Premanathan, K. Karthikeyan, K. Jeyasubramanian, G. Manivannan, Selective tox-
icity of ZnO nanoparticles toward gram positive bacteria and cancer cells by apopto-
sis through lipid peroxidation, Nanomedicine 7 (2011) 184–192.
[14] G. Floresa, J. Carrilloa, J.A. Lunaa, R. Martinezb, A. Sierra-Fernandezc, O. Milosevicd,
M.E. Rabanale, Synthesis, characterization and photocatalytic properties of nano-
structured ZnO particles obtained by low temperature air-assisted-USP, Adv. Pow-
der Technol. 25 (2014) 1435–1441.
[15] M. Ahmad, E. Ahmed, Fezza Zafar, N.R. Khalid, N.A. Niaz, Abdul Hafeez, M. Ikram, M.
Ajmal Khan, Zhanglian Hong, Enhanced photocatalytic activity of Ce-doped ZnO
nanopowders synthesized by combustion method, J. Rare Earths 33 (2015)
255–262.
[16] Alireza Khataee Atefeh Karimi, Samira Arefi-Oskoui, Reza Darvishi Cheshmeh
Soltani, Younes Hanifehpour, Behzad Soltani, Sang Woo Joo, Sonochemical synthesis
of Pr-doped ZnO nanoparticles for sonocatalytic degradation of Acid Red 17,
Ultrason. Sonochem. 22 (2015) 371–381.
[17] Xiu Ming Ren, He Qiu Zhang, Li Zhong Hu, Jiu Yu Ji, Yang Li, Jun Lin Liu, Hong Wei
Liang, Ying Ming Luo, Ji Ming Bian, The effect of growth time on the morphology
of ZnO nanorods by hydrothermal method, Adv. Mater. Res. (2013) 855–859.
[18] Tan Lin, Lihomng Wang, Yude Wang, Hydrothermal synthesis of SnO2 nanostruc-
tures with different morphologies and their optical properties, J. Nanomater. 23
(2011).
[19] A. Moulahi, F. Sediri, Pencil-like zinc oxide micro/nano-scale structures: hydrother-
mal synthesis, optical and photocatalytic properties, Mater. Res. Bull. 48 (2013)
3723–3728.
[20] Dhaneswar Das, Bikash Chandra Nath, Pinkee Phukon, Amarjyoti Kalita, Swapan
Kumar Dolui, Synthesis of ZnO nanoparticles and evaluation of antioxidant and cy-
totoxic activity, Colloids Surf. B: Biointerfaces 111 (2013) 556–560.
[21] W. Brand Williams, Use of a free radical method to evaluate antioxidant activity,
Food Sci. Technol. 28 (1995) 23–30.
[22] P.C. Nethravathi, G.S. Shruthi, D. Suresh, Udayabhanu, H. Nagabhushana, S.C.
Sharma, Garcinia xanthochymus mediated green synthesis of ZnO nanoparti-
cles: Photoluminescence, photocatatlytic and antioxidant studies, Ceram. Int.
41 (2015) 8680–8687.
[23] A. Mishra, N. Chaudhary, Study of povidone iodine loaded hydrogels as wound
dressing material, trends biomater, Artif. Organs 23 (2010) 122.
[24] S.P.M. Crouch, R. Kozlowski, K.J. Slater, J. Fletcher, The use of ATP bioluminescence as
a measure of cell proliferation and cytotoxcity, J. Immunol. Methods 160 (1993)
81–88.
[25] R.J. Gonzalez, J.B. Tarloff, Evaluation of hepatic sub cellular fractions for alamar blue
and MTT reductase activity, Toxicol. in Vitro 15 (2001) 257–259.
[26] N. Hattori, T. Sakakibara, N. Kajiyama, T. Igarashi, M. Maeda, S. Murakami, Enhanced
microbial biomass assay using mutant luciferase resistant to benzalkonium chloride,
Anal. Biochem. 319 (2003) 287–295.
[27] L. Kangas, M. Gronroos, A.L. Nieminen, Bioluminescence of cellular ATP: a new
method for evaluating cytotoxic agents in vitro, Med. Biol. 62 (1984) 338–343.
[28] A. Lundin, M. Hasenson, J. Persson, A. Pousette, Estimation of biomass in growing
cell lines by adenosine triphosphate assay, Methods Enzymol. 133 (1986) 27–42.
[29] F.F. Denizot, R.R. Lang, Rapid colorimetric assay for cell growth and survival, modi-
fications to the tetrazolium dye procedure giving improved sensitivity and reliabil-
ity, J. Immunol. Methods 22 (1986) 271–277.
[30] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63.
[31] Ameer Azam, A.S. Ahmed, Mohammad Oves, M.S. Khan, S.S. Habib, Adnan Memic,
Antimicrobial activity of metal oxide nanoparticles against Gram-positive and
Gram-negative bacteria: a comparative study, Int. J. Nanomedicine 7 (2012)
6003–6009.
[32] J. Zhang, L. Sun, J. Yin, H. Su, C. Liao, Control of ZnO morphology via a simple solution
route, Chem. Mater. 14 (2002) 4172–4177.
[33] Q. Ahsanulhaq, A. Umar, Y.B. Hahn, Growth of aligned ZnO nanorods and
nanopencils on ZnO/Si in aqueous solution: growth mechanism and structural
and optical properties, Nanotechnology 18 (2007) 115603.
[34] A.I.Y. Tok, F.Y.C. Boey, S.W. Du, B.K. Wong, Flame spray synthesis of ZrO2 nano-
particles using liquid precursor, Mater. Sci. Eng. 130 (2006) 114–119.
[35] M. Xiangyang, Z. Hui, J. Yujie, X. Jin, Y. Deren, Sequential occurrence of ZnO
nanopaticles, nanorods and nanotips during hydrothermal process in a dilute aque-
ous solution, Mater. Lett. 59 (2005) 3393–3397.
[36] P.M. Aneesh, K.A. Vanaja, M.K. Jayaraj, Synthesis of ZnO nanoparticles by hydrother-
mal method, Nanophotonic Mater. 6639 (2007).
[37] M. Heinlaan, A. Ivask, I. Blinova, H.C. Dubourguier, A. Kahru, Toxicity of nanosized
and bulk ZnO, CuO and TiO2 to bacteria vibrio fischeri and crustaceans Daphnia
magna and Thamnocephalus platyurus, Chemosphere 71 (2008) 1308–1316.
[38] H. Yang, C. Liu, D. Yang, H. Zhang, Z. Xi, Comparative study of cytotoxicity, oxidative
stress and genotoxicity induced by four typical nanomaterials: the role of particle
size, shape and composition, J. Appl. Toxicol. 29 (2009) 69–78.
[39] O. Akhavan, M. Mehrabian, K. Mirabbaszadeh, R. Azimirad, Hydrothermal syn-
thesis of ZnO nanorod arrays for photocatalytic inactivation of bacteria, J. Phys.
D. Appl. Phys. 42 (2009).
[40] N.M. Franklin, N.J. Rogers, S.C. Apte, G.E. Batley, G.E. Gadd, P.S. Casey, Comparative
toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga
(Pseudokirchneriella subcapitata): the importance of particle solubility, Environ. Sci.
Technol. 41 (2007) 8484–8490.
[41] T. Xu, C.S. Xie, Tetrapod-like nano-particle ZnO/acrylic resin composite and its
multi-function property, Prog. Org. Coat. 46 (2003) 297–301.
[42] L.L. Zhang, Y.H. Jiang, Y.L. Ding, M. Povey, D. York, Investigations into the antibacte-
rial behavior of suspensions of ZnO nanoparticles (ZnO nanofluids), J. Nanoparticles
9 (2007) 479–489.
[43] Cory Hanley, Janet Layne, Alex Punnoose, K.M. Reddy, Isaac Coombs, Andrew
Coombs, Kevin Feris, Denise Wingett, Preferential killing of cancer cells and activat-
ed human T cells using ZnO nanoparticles, Nanotechnology 19 (2008), 295103.