Materials Science and Engineering C 79 (2017) 465472

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Materials Science and Engineering C

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

Functionalization of ZnO nanoparticles by 3-mercaptopropionic acid for

aqueous curcumin delivery: Synthesis, characterization, and
anticancer assessment
Seyed-Behnam Ghaffari a, Mohammad-Hossein Sarrafzadeh a,, Zahra Fakhroueian a,
Shadab Shahriari b, M. Reza Khorramizadeh b,c,
a
School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran
b
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
c
Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

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

Article history: Inherent biocompatibility and stability of zinc oxide nanoparticles (ZnO-NPs) and their biomedical potentials
Received 4 December 2016 make them an emerging candidate for drug delivery. The aim of this study was to develop and assess a simple
Received in revised form 1 May 2017 procedure for surface functionalization of ZnO-NPs by 3-mercaptopropionic acid (MPA) for water-soluble
Accepted 10 May 2017
curcumin delivery. Carboxyl-terminated ZnO nanoparticles were successfully made using ZnCl2 and NaOH in
Available online 11 May 2017
the presence of MPA. The functional groups were activated by 1,1-carbonyldiimidazole (CDI) and the curcumin
Keywords:
bonding was carried out at room temperature for 24 h. The core-shell nanocomposite had a signicant better sol-
ZnO nanoparticles ubility versus free curcumin, as characterized by XRD, FTIR, UVVis spectrophotometry, DLS, and TEM, p b 0.005.
Surface functionalization In addition, MTT cytotoxicity assessment on MDA-MB-231 breast cancer cells revealed a drop of IC50 values from
3-Mercaptopropionic acid 5 g/mL to 3.3 g/mL for free curcumin and ZnO-MPA-curcumin complex, respectively. This result showed an
Curcumin augmented cancer-inhibitory effect of nanoconjugate complex. In conclusion, the presented improved solubility
Anticancer agent and elevated functionality of novel ZnO-MPA-curcumin nanoformula is promising, and could be considered for
new therapeutic endeavors.
2017 Elsevier B.V. All rights reserved.

1. Introduction Identication and manipulation of the parameters by which ZnO-

The remarkable high surface area, appropriate biocompatibility, low
toxicity and chemical stability characteristics of zinc oxide nanoparticles
(ZnO-NPs) have attracted increasing recent biomedical investigations.
Bearing unique physicochemical properties, i.e. piezoelectric, catalytic
and optic features [1], ZnO-NPs has been studied for biosensing [2], im-
aging [3] and antibacterial [4] applications, as well.
Hanley and co-workers have initially indicated a selective capability
of ZnO nanoparticles to destruct cancerous T cells, escaping normal cells
[5]. More reports on inherent cancer invading functions, bioimaging,
and photodynamic therapy (PDT) properties of ZnO nanostructures
have been cited ever since [610].
Corresponding author.
Correspondence to: M. Reza Khorramizadeh, Endocrinology and Metabolism Research
Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of
Medical Sciences, Tehran, Iran.
E-mail addresses: Sarrafzdh@ut.ac.ir (M.-H. Sarrafzadeh), khoramza@tums.ac.ir
(M.R. Khorramizadeh).
NPs contribute destruction of tumor cells maintain customizing and im-
proving the potential biomedical applications of these nanoparticles.
Particle size, surface charge, shape, solubility and surface chemistry
are main physicochemical characteristics that have been showed to
play critical role in determining biological behaviors of nanoparticles
[1113].
It has been reported that ZnO-NPs can readily agglomerate, owing to
their high surface energy [14]. Therefore, having an applicable strategy
to control ZnO-NPs agglomeration, e.g. surfactant coating or capping,
is crucial to encounter biouids. In addition, for such biological applica-
tions as sensing or drug delivery, the surface of ZnO-NPs should be func-
tionalized to preserve water solubility and probe/drugs bioconjugate
attachment. Although, bulkier form of ZnO particles have been recog-
nized as generally recognized as safe (GRAS) material by the FDA and
many researchers have indicated the low toxicity of their nano-scale
form against normal cells, coating of nanoparticles is also an appropriate
method for reducing unwanted toxicity and improving biocompatibility
of nanoparticles [8]. To date, a number of capping agents or polymeric
compounds have been employed to modify and functionalize
ZnO nanoparticles: 3-aminopropyltriethoxysilane (APTS) [15],

http://dx.doi.org/10.1016/j.msec.2017.05.065
0928-4931/ 2017 Elsevier B.V. All rights reserved.
466 S.-B. Ghaffari et al. / Materials Science and Engineering C 79 (2017) 465472
monoethanolamine (MEA) [16], mercaptoundecanoic acid (MUA) [17],
mercaptoacetic acid (MAA) [18] and fatty acid [19], respectively.
Curcumin, an active ingredient of turmeric, has been extensively in-
vestigated in the treatment of various illnesses, including cancer, cardio-
vascular disease, inammatory bowel, wound healing, Alzheimer's dis-
ease, rheumatoid arthritis, and diabetes [20]. However, low water solu-
bility and bioavailability, rapid metabolization, and poor chemical
stability have rendered utilization of curcumin limited in clinical trials
[21]. Curcumin was reported undetectable when its daily oral adminis-
tration was b 8 g [22].
To overcome these problems, various types of curcumin
nanoformulations, usually referred to as curcumin-loaded organic sys-
tems have been developed: solid lipid nanoparticles, micelles, lipo-
somes, nanogels, cyclodextrins, and dendrimers [23]. In line with the
increasing applications of metal and metal oxide nanoparticles as
nanocarriers, researchers have developed nanoformulations in which
curcumin molecules are bound to gold [24], iron oxide [25], silica [26],
and recently, zinc oxide [27] nanoparticles. These systems demonstrate
advantages as easily tailored for favorable therapeutic purposes.
In this study, a novel ZnO-NPs functionalization procedure, using 3-
mercaptopropionic acid, was developed for curcumin conjugation in a
water-soluble delivery system. This nanoformulation can enhance
curcumin solubility and stability. The enhanced bioavailability, together
with inherent preferential tumor toxicity of ZnO-NPs, eventually im-
proves therapeutic applicability of curcumin. Several characterization
techniques were performed to analyze the physicochemical properties of
this engineered core-shell nanocomposite and its anticancer activity on
MDA-MB-231 breast cancer cell lines has been evaluated by MTT assay.
2. Material and methods
2.1. Chemicals, cell line and initial cell culture
Zinc chloride, Sodium hydroxide, 3-mercaptopropionic acid, 1,1-
carbonyldiimidazole (CDI) and curcumin were purchased from Merck.
Dried dimethylsulphoxide (DMSO) and ethanol were obtained from
Ameretat Shimi, Iran.
MDA-MB-231 breast cancer cells lines (German Collection of Micro-
organisms and Cell Cultures, DSMZ, Braunschweig, Germany) were
grown in Dulbecco's modied Eagle's medium (DMEM) supplemented
with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin in
a humidied 5% CO2 incubator at 37 C. Cells were kept at this condition
prior to MTT cytotoxicity assay.
2.2. Synthesis of 3-mercaptopropionic acid-capped ZnO
Zinc oxide nanoparticles were produced by a simple co-precipitation
route with zinc chloride as the zinc source and NaOH as the precipitation
agent. Surface modication and functionalization of ZnO nanoparticles
carried out with 3-mercaptopropionic acid (MPA) during the ZnO-NPs
formation. 2 g of the zinc chloride was dissolved in 60 mL of water-etha-
nol (1:1) solution. Then 0.2 mL of MPA was added drop by drop to the so-
lution under stirring condition. Subsequently, 2 M NaOH aqueous solution
was slowly added to the mixture until the pH value was adjusted to 11.
This solution was reuxed for 24 h at room temperature. The precipitates
washed repeatedly with a mixture of distilled water and ethanol and
dried in an oven at 70 C overnight. Unmodied-zinc oxide particles
were produced by the same procedure without MPA reagent.
2.3. Covalent conjugation of curcumin to the ZnO-MPA nanoparticles
The modied zinc oxide nanoparticles were re-dispersed in dry
DMSO and then 0.5 g 1,1-carbonyldiimidazole (CDI) was added to the
suspension for activating the carboxyl groups on the surface of ZnO
nanoparticles. After reuxing for 2 h, the activated powders were isolat-
ed and re-dispersed in DMSO. Subsequently, 0.2 g curcumin was added
to the solution. The mixture was continuously shaken for 24 h at room
temperature. Obtained product (The conjugate product) separated by
centrifugation, washed repeatedly with DMSO to remove unattached
curcumin and nally dried at 60 C overnight.
2.4. Characterization of products
The phase characterization of as-synthesized materials was obtained
by an X-ray diffractometer (XRD; Siemens D-500). The analysis was
done in the 2 range from 10 to 90 using copper K1 ( = 1.54056
) radiation. The crystallite sizes of the products were calculated by
Scherer's formula [28].
The particle size and morphology of the products was investigated
using a transmission electron microscope (TEM; Zeiss-EM10C-100 KV)
and a eld-emission scanning electron microscopy equipped with an
EDAX energy dispersive X-ray analyzer (SEM; Zeiss-SIGMA VP-500).
The hydrodynamic size and zeta potential of the prepared particles
were measured with Dynamic light scattering (DLS; ZetaPlus,
Brookhaven Instruments). The procedure of DLS samples preparation
included a dispersion of nanoparticles in distilled water (1 mg/mL)
and subsequently sonication for 5 min (bath sonication). To investigate
the effect of serum on the hydrodynamic size of the conjugate product,
the complex dispersed rst in DMED + 10% FBS (1 mg/mL) and stirred
for 3 h and then re-dispersed in water.
Fourier Transform Infrared (FTIR) spectra were recorded with Perkin
Elmer FT-R Spectrometer, using KBr pellets, in the region of 400
4000 cm 1. Ultravioletvisible (UVVis) spectra were recorded by
OPTIZEN 2120UV plus spectrophotometer in the range of 200600 nm.
2.5. Estimation of curcumin content in the conjugate product
Standard calibration curve of curcumin in DMSO was obtained from
the absorption intensity by UVVis spectroscopy at 430 nm. The con-
centration of curcumin in the conjugate product was estimated by mea-
suring the present of free curcumin (non-attached curcumin) in the
supernatant of synthesis procedure of ZnO-MPA-curcumin. The amount
of attached curcumin was calculated using the formula:
Amount of curcumin conjugated = Total amount of curcumin used
the amount of curcumin in the supernatant.
2.6. Sedimentation study
The sedimentation rate of ZnO-MPA-curcumin particles was investi-
gated by tracking the optical absorbance at 400 nm as a function of time,
using UVVis spectrophotometry.
2.7. In vitro cytotoxicity assay
An optimized MTT reagent-based colorimetric assay was carried out
to assess possible cytotoxic effects of native curcumin, ZnO-MPA NPs
and ZnO-MPA-curcumin reagents, respectively [29]. Briey, MDA-MB-
231 breast cancer cells were plated into 96 well plates at a density of
10,000 cells/well in 200 L complete culture medium and allowed to at-
tach overnight. Cells in the exponential phase of growth, were treated
with desired concentrations of the above-named treatment reagents
for 48 h. Dispersion of the samples in cell culture was down using a
bath sonicator for 5 min. After treatment, cells were washed twice
with PBS and then 20 L of MTT (3-(4,5-dimethylthiazolyl)-2,5-diphe-
nyltetrazolium bromide) (Sigma-Aldrich) dissolved in PBS (4 mg/mL)
was added directly to all the wells, and the plates were incubated for
3 h at 37 C. After removal of the supernatant, the formazan crystals
that formed were dissolved by addition of 60 L DMSO per well. The
color intensity resulting from the formation of soluble formazan prod-
uct was measured at 570 nm with 660 nm set as the reference wave-
length using a microplate reader (Anthos, Austria). This absorption
reading is directly proportional to the number of live cells in the culture.
 S.-B. Ghaffari et al. / Materials Science and Engineering C 79 (2017) 465472
Fig. 1. XRD patterns of ZnO, ZnO-MPA, and ZnO-MPA-curcumin powders.
The percentage of viability was presented as the percentage of the ab-
sorption of treated cells to the absorption of untreated cells as the neg-
ative control. The maximum DMSO percentage as the solvent of
curcumin in cell culture was 0.5% (v/v). DMSO 0.5% showed an absor-
bance value similar to control indicating that DMSO 0.5% have no effect
on the cells. Data presented are from three replicates.
3. Results and discussion
3.1. ZnO functionalization by 3-mercaptopropionic acid
Zinc oxide nanoparticles, either alone or loaded with biological
probes, are considered as novel compounds for cancer treatment and
Fig. 2. FTIR spectra of ZnO, ZnO-MPA, ZnO-MPA-curcumin and free curcumin.
467
biosensing researches. One applicable approach to enhance biomedical
properties, could be conjugation of monodispersed ZnO-NPs with simi-
lar-effect biomolecules. Various methods have been developed to
functionalize ZnO-NPs and improve their water solubility and size sta-
bility. In the present study, a simple chemical approach was designed
to produce functionalized zinc oxide nanoparticles from zinc chloride
solution using MPA as a capping agent containing a carboxyl group.
This functional group can provide improved NPs aqueous-solubility
and enhanced linkage to biological molecules which usually contain dif-
ferent functional groups like amide or hydroxyl moieties.
The formation mechanism of zinc oxide crystals in the chloride solu-
tions was comprehensively discussed elsewhere [30]. To verify forma-
tion of ZnO phase and study the inuence of the surface
functionalization on the crystallinity of ZnO-NPs, XRD analysis was car-
ried out. Fig. 1 shows the XRD pattern of this products. All the diffraction
peaks in the diffraction patterns of the powders obtained from ZnO,
ZnO-MPA synthesis procedures showed in accordance with the JCPDS
prole of hexagonal wurtzite zinc oxide (JCPDS #36-1451). Since no sig-
nicant difference has been seen between the two patterns, it can be
concluded that the surface modication did not change the crystalline
structure of the zinc oxide nanoparticles. The crystallite size of zinc
oxide tends to be smaller in the presence of MPA molecules. This
value for ZnO and ZnO-MPA particles were 62 nm and 23 nm,
respectively.
FTIR spectroscopy was used to investigate the incorporation of MPA
to the surface of ZnO-NPs. Fig. 2 displays FTIR spectra of as-prepared
powders along with free curcumin. FTIR of all samples clearly showed
a broad peak between 3600 and 3050 cm1 and intense peaks between
420 and 500 cm 1 reecting the presence of hydroxyl groups and
Zn\\O bonds, respectively. The peak at 1398 cm 1 in ZnO and ZnO-
MPA is related to the C\\OH bonding. This peak was shifted to
1411 cm1 in the conjugate product. The sharp peak at 1569 cm1 in
the spectrum of ZnO-MPA implied the presence of COOH groups on
the surface of the modied ZnO-NPs [14]. Two chemical procedures
are probably involved in the formation of chemical bonds between
zinc oxide and the MPA molecules. The First procedure is dehydration
reaction between carboxyl groups in MPA and hydroxyl groups at the
surface of ZnO crystals. Secondly, is the replacement of the oxygen
468 S.-B. Ghaffari et al. / Materials Science and Engineering C 79 (2017) 465472
atoms in ZnO crystals by S atoms of MPA molecules [17]. The absence of
a peak at 2550 cm1 related to SH bond most likely indicates the forma-
tion of ZnO-MPA complex by the substitution reaction.
Fig. 3 shows typical TEM and SEM images and the corresponding
EDS spectra of ZnO-MPA particles. Based on both microscopic images,
the particles have semi-spherical morphology. The core/shell structure
of the particles can also be seen in the TEM image where the core and
the outer layer in particles are clearly distinguishable. Based on the
TEM images of N 100 particles, the average diameter size of ZnO-MPA
particles was about 38 nm. The EDS characterization revealed propor-
tions of 61.6% zinc, 17% oxygen, 20.7% carbon, and 0.8% sulfur and indi-
cated the presence of MPA elements in the complex.
Another technique to approve the binding of MPA to ZnO
nanocrystals and to verify the presence of carboxyl groups is to compare
the aqueous zeta potential of ZnO-MPA with bare ZnO particles. The
point of zero charge (PZC) of ZnO is 8.410.3. This indicates that the
charge of ZnO particles is positive in water and nutrient pH buffers. As
compared with the zeta potential of bare ZnO particles (+12 mV), the
surface charge of the modied ZnO nanoparticles (calculated by DLS)
was 31 mV that could be associated with the negative charge of the
carboxyl groups in water (Table 1). The calculated charge-differences
is indicative that MPA has been bonded to the zinc oxide nanoparticles
surface. The strong negative charge of the modied nanoparticles
caused a reduction of the agglomeration of particles. Accordingly, the
Fig. 3. (a) TEM image of ZnO-MPA particles showing the core/shell structure of nanoparticles. (b) SEM image and (c) the corresponding EDS spectra of ZnO-MPA particles.
hydrodynamic size of nanoparticles decreased from 431 nm to 40 nm.
This value is very close to the mean diameter size obtained by TEM
images.
The UVVisible light absorption spectra of ZnO particles and MPA-
capped ZnO-NPs are illustrated in Fig. 4. ZnO and ZnO-MPA NPs exhib-
ited their characteristic strong absorption bands corresponding to bend-
edge absorption at 354 and 342 nm, respectively. This blue shift of the
absorption edge in ZnO-MPA NPs compared with non-modied ZnO
could be attributed to a quantum connement effect, probably due to
the smaller size and less agglomeration of the modied ZnO-NPs.
3.2. Curcumin bonding to MPA capped ZnO-NPs
The direct conversion of a carboxylic acid to an ester with hydroxyl
groups on the curcumin molecule is a very complicated process. In con-
trast, formation of ester moiety would be much easier, if the carboxylic
groups are rst activated. 1,1-carbonyldiimidazole (CDI) is a highly ef-
cient activating-agent for carboxylic acids. Moreover, fewer side reac-
tions are associated with CDI, as compared to other alternative
molecules like dicyclohexylcarbodiimide (DCC). The activation proce-
dure was performed in DMSO, as DMSO is a suitable solvent for
curcumin. During the process, a reactive intermediate, N-acylimidazole,
is generated which can then react with hydroxyl groups in curcumin to
form ester linkages [31].
 S.-B. Ghaffari et al. / Materials Science and Engineering C 79 (2017) 465472 469

Table 1 spectrum of curcumin showed a strong maximum at 430 nm. The spec-
Hydrodynamic sizes and zeta potentials of the samples. trum of ZnO-MPA-curcumin exhibited two absorption maximums at
Sample Hydrodynamic size (nm) Polydispersity Zeta potential 348 and 425 nm, respectively. The presence of the peak at 425 nm, relat-
in water index (mV) ed to the adsorption behavior of curcumin, clearly indicated that the
ZnO 431 0.35 +12 drug adsorbed well on the surface of ZnO-MPA nanoparticles. The
ZnO-MPA 40 nm 0.12 31 mere 5 nm blue shift in the maximum wavelength peak was derived
ZnO-MPA-curcumin 904 nm 0.243 15 from the reaction of hydroxyl groups with the activated carboxyl groups
157 nm in DMEM + 10% 0.176
and the formation of chemical bonds between MPA and curcumin. The
FBS
active concentration of curcumin in the complex was estimated about
130 g per mg of the conjugate (about 13% w:w of the complex).
Shape and size particle of the conjugate product were studied by
FTIR spectrum of curcumin showed peaks at: i)1628 cm1, indicat- SEM-EDS, TEM, and dynamic light scattering (DLS). The EDS character-
ing C_C symmetric aromatic ring stretching; ii)1508 cm1 contributed ization of the particles (Fig. 5a) revealed proportions of 46% zinc, 18.4%
to C_O of benzene ring; iii)1427 cm1 associated with C\\H bending oxygen, 35.2% carbon, and 0.4% sulfur. A relatively higher proportion of
vibration; iv)1279 cm 1 represents aromatic C\\O stretching vibra- carbon was indicative of curcumin molecules bindings. The SEM image
tions; and v)961 cm1 related to C\\O\\C stretching vibrations (Fig. shows the semi-spherical morphology of particles (Fig. 5b). Core-shell
2). FTIR spectrum of ZnO-MPA-curcumin conjugate demonstrated and a multi-layer structure of the ZnO-MPA-curcumin formulation
peaks related to ZnO-MPA. In addition, curcumin-related main peaks, and its hypothetical schematic were demonstrated in TEM images
especially the peak at 1490 cm1, is assigned to C_O of aromatic (Fig. 5c, d and e) where ZnO is the core and MPA and curcumin mole-
rings of curcumin. The sharp peak at 1090 cm1 clearly indicated the es- cules are positioned as the second and third layers, respectively. The av-
terication of the carboxyl groups and the formation of the ester linkage erage diameter size of the core was about 74 nm and the mean diameter
(C\\O\\C). These results conrm the covalent attachment of curcumin of the total complex was about 121 nm. Therefore, this core-shell struc-
to the surface of ZnO-MPA NPs. ture can be concluded as a nanocomposite. The size of ZnO-MPA area is
XRD pattern of the conjugate product observed in Fig. 1 has no obvi- larger, as compared with TEM image in Fig. 3. This size discrepancy de-
ous differences with the pattern of ZnO-MPA NPs. This indicates the rived from probable agglomeration of ZnO-MPA particles in the activa-
amorphous nature of curcumin, bonded to the surface of modied tion step.
ZnO-NPs. Crystallite size of ZnO in the conjugate product was 55 nm. The size of nanoformulations in body uids is very important for
In the present study, the light absorption behavior of free curcumin drug delivery purposes. The behavior of the particles was therefore in-
has been analyzed in methanol. Curcumin has an intense absorption be- vestigated in water and in serum-containing media (simulating blood
tween the wavelength of 340535 nm, depending on the solvent char- plasma condition). As shown in Table 1, the hydrodynamic size of the
acteristic and its structural modication [32]. According to Fig. 4, the conjugate product, when dispersed in de-ionized water, was

Fig. 4. UVVisible spectra of (a) ZnO and ZnO-MPA NPs and (b) ZnO-MPA-curcumin and free curcumin.
470 S.-B. Ghaffari et al. / Materials Science and Engineering C 79 (2017) 465472

Fig. 5. (a) EDS spectra and (b) SEM image of the conjugate product. (c) and (d) TEM images of ZnO-MPA-curcumin nanocomposite and (e) its hypothetical schematic.

approximately 904 nm, depicting possible aggregated curcumin-coated
ZnO-MPA nanoparticles. This aggregation could be resulted from hydro-
phobic nature of curcumin and strong reduction of the zeta potential of
the particles (shifting from 15 mV to 31 mV). The agglomeration of
the conjugate particles was decreased clearly to 157 nm when they
were initially suspended in cell culture medium supplemented with
serum (DMEM + 10% FBS) and then, re-dispersed in water. The amelio-
ration of agglomerated-NPs problem could presumably be due to
protein adsorption on the surfaces of particles [33]. This hydrodynamic
size (157 nm) has been reported as an effective size for the purpose of
passive delivery to tumor cells (b~200 nm but N~10 nm), as well [34].
Furthermore, particle sedimentation rate of the conjugate product
showed dependence on the suspending media. Fig. 6 represented the
sedimentation rate of ZnO-MPA-curcumin nanocomposite in de-ion-
ized water and in the cell culture medium containing 10% serum. The
particles in water displayed a faster deposition rate than those in the
 S.-B. Ghaffari et al. / Materials Science and Engineering C 79 (2017) 465472 471

Fig. 6. Sedimentation curves of ZnO-MPA-curcumin particles in water and DMEM medium + 10% FBS, vertical axis declared with regard to optical absorbance (using UVVis
spectrophotometry at 400 nm) at time t (At) relative to the value at t = 0 (A0).

cell culture medium. Almost 40% of the particles precipitated within
24 h in water. Conversely, free suspended curcumin precipitated imme-
diately in water. However, the conjugate particles could extremely dis-
perse better. The sedimentation rate was signicantly slower in the cell
culture medium with serum (only 8% of the particles deposited in the
same time period). Therefore, pre-dispersion of the conjugate particles
in serum-containing solution can be a stabilizing method and a suitable
technique for biological studies.
3.3. Anticancer potential of the conjugate product
Conjugation of anticancer drug molecules may alter their functional-
ity. It is therefore imperative to re-assess the anticancer properties of
the complexed molecules after the conjugation process. Cytotoxicity
tests are employed as the bottom line analyses, accordingly. MTT
assay revealed curcumin, ZnO-MPA nanoparticles, and the conjugate
product cytotoxic effects on MDA-MB-231 breast cancer cell model in
a dose-dependent manner, respectively (Fig. 7).
When cells were treated with curcumin, the viability of the can-
cer cells decreased sharply in a dose-dependent manner. A similar
pattern could be seen with ZnO-MPA and ZnO-MPA-curcumin at all
tested concentrations. The half maximal inhibitory concentration
(IC50: 50% cell growth inhibitory concentration) was calculated as 5
g/mL (curcumin), 38 g/mL (ZnO-MPA), and 25 g/mL (ZnO-MPA-
curcumin), respectively. IC50 value of the conjugated molecules, cal-
culated from curcumin concentration base curve, was about 3.3 g/
mL which is higher than that of free curcumin. This interesting nd-
ing revealed the augmented cancer inhibitory effects of conjugated
molecules, as compared to free curcumin. In other words, anticancer
property of ZnO nanoparticles has been preserved during the
nanoformulation and conjugation methodology presented in this
study.

Fig. 7. Viability of MDA-MB-231 cells (MTT assay) after 48 h exposure to (a) free curcumin (b) ZnO-MPA NPs (c) ZnO-MPA-curcumin nanocomposite and (d) ZnO-MPA-curcumin
nanocomposite based on the concentration of curcumin in the complex. Data represented as the means SD of three identical experiments derived from three replicate.
472 S.-B. Ghaffari et al. / Materials Science and Engineering C 79 (2017) 465472
4. Conclusions
In this study, ZnO-NPs were successfully functionalized via a novel
chemical procedure using 3-mercaptopropionic acid (MPA). This moie-
ty was covalently conjugated to curcumin. The synthesized conjugate
product was then characterized, and its cytotoxicity was assessed
using MDA-MB-231 breast cancer cell model. Considering the inherent
anticancer effect of zinc oxide and the capability of functionalized to at-
tach drugs makes ZnO-NPs favorable for drug delivery applications. The
MPA functionalization procedure presented in this study might be
amply customized to form targeted drug delivery endeavors. Curcumin,
as a natural molecule with several therapeutic potentials, primarily suf-
fers from low solubility and bioavailability in its free molecule form, was
covalently conjugated to the surface of as-synthesized ZnO-MPA NPs.
Physicochemical properties of the nanocomposite were evaluated
with several techniques. Our results indicated that the nanocomposite
had a core-shell structure and can be dispersed well in cell culture me-
dium in the presence of serum. MTT assay indicated that not only the
chemical binding of curcumin molecules to ZnO particles did not reduce
the anticancer activity of curcumin, but the conjugate product had also
higher cytotoxicity against the tumor cells. The conjugate product could
bear several potentials for cancer treatment such as chemotherapy,
photodynamic therapy (PDT) and imaging. However, its commerciali-
zation needs a series of in-vitro and in-vivo analyses that are presently
in progress at our lab.
Acknowledgment
The authors gratefully acknowledge the nancial support provided
by Iran National Science Foundation (INSF) (Grant No. 92025964).
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