Author’s Accepted Manuscript

Novel synthesis of ZnO Nanoparticles and their

Enhanced Anticancer activity: Role of ZnO as a
drug carrier

Mohamed F. Al-Ajmi, Afzal Hussain, Faheem

Ahmed
www.elsevier.com/locate/ceri

PII: S0272-8842(15)02238-5
DOI: http://dx.doi.org/10.1016/j.ceramint.2015.11.133
Reference: CERI11750
To appear in: Ceramics International
Received date: 11 November 2015
Revised date: 22 November 2015
Accepted date: 23 November 2015
Cite this article as: Mohamed F. Al-Ajmi, Afzal Hussain and Faheem Ahmed,
Novel synthesis of ZnO Nanoparticles and their Enhanced Anticancer activity:
Role of ZnO as a drug carrier, Ceramics International,
http://dx.doi.org/10.1016/j.ceramint.2015.11.133
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 Novel synthesis of ZnO Nanoparticles and their Enhanced Anticancer activity:
Role of ZnO as a drug carrier

Mohamed F. Al-Ajmi1, Afzal Hussain1*, Faheem Ahmed2*

1
Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia1

2
College of Science and General studies, Alfaisal University, P.O. Box 50927, Riyadh 11533,
Saudi Arabia

Abstract

In this work, a simple and versatile technique was developed to prepare highly crystalline ZnO
nanoparticles (ZnO NPs) by organic precursor method using 5, 6 dimethyl benzimidazole and
Zn(CH3COO)2.2H2O followed by calcination. These synthesized ZnO NPs were used as a drug
carrier to form 5-Fluorouracil (5 Fu) encapsulated ZnO NPs by varying the molar ratio (100-
300:1) of ZnO NPs to 5-Fu. X-ray diffraction (XRD) results indicated that the ZnO NPs had
single phase nature with the wurtzite structure. Field emission scanning electron microscopy
(FESEM) and Transmission electron microscopy (TEM) results showed nanometer dimension of
the NPs. FTIR analysis further reaffirmed the formation/Encapsulation of ZnO NPs. UV-Vis
spectroscopy determined the encapsulation efficiency (EE) and loading capacity (LC) of 5-Fu
drug on ZnO NPs. HPLC analysis of encapsulated NPs indicated release of 5-Fu was higher at
tumor cell pH (pH 6.0) than physiological pH. Moreover, the Anti-tumor activity of ZnO NPs
and 5-Fu-encapsulated ZnO NPs investigated using flow cytometry demonstrated that 5-Fu
encapsulated ZnO NPs have more anti-tumor activities than 5-Fu itself toward MCF-7 (Breast
cancer) cell line. Also, cytotoxicity of MCF-7 increased with the increase of ZnO NPs: 5-Fu
ratio. This research will introduce a new concept to synthesize 5-fluorouracil encapsulated ZnO
NPs and its application towards the cancer cell line. Thus, the ZnO NPs could not only apply as
the drug carrier to deliver 5-Fluorouracil into the cancer cells, but also enhances anti-tumor
activity.
Keywords: Flow Cytometry, ZnO NPs, MCF-7, Encapsulation.
*Corresponding Email: fahahmed@alfaisal.edu; Phone: +966-2137741; Fax: +966-2137741
1. Introduction

In spite of some medications, millions of peoples are dying every year due to cancer.

Additionally, the survival patients suffer from various serious side effects due to the use of

available antineoplastic medicines. The development of nanoparticles based drugs seems to be

effective providing little side effects and targeted action on cancer cells due to the large surface

area of nanoparticles that facilitate the incorporation of high drug doses [1-3]. Various types of

nano-sized drug carriers, such as liposomes, polymeric micelles, dendrimers, and inorganic NPs

have been investigated in cancer therapy in order to minimize the side effects of anticancer drugs

and enhance the antitumor drug efficacy target therapies [4,5]. Inorganic NPs, including metal

and metal oxides (gold, nickel, silver, iron oxide, zinc oxide, gadolinium, and titanium dioxide

particles), are promising materials for applications in medicine, such as cell imaging, biosensing,

drug/gene delivery, and cancer therapy [1-3, 6-8]. Even though, metal and metal oxide

nanoparticles are biocompatible and inert vehicles, a significant fraction of metal/metal oxide

particles can be retained and accumulated in the body after drug administration, possibly causing

toxicity [9]. Among these nanoparticles, Zinc oxide (ZnO) NPs have received much attention for

their implications for cancer therapy [10] and has various advantages such as it exhibits

anticancer activity at physiological pH. The anticancer activity of ZnO is also considered due to

the generation of active oxide, hydrogen peroxide (H2O2) and superoxide, produced from its

surface [11]. Furthermore, there has been growing interest in semiconductor nanomaterial as

anticancer drug carriers very recently, which represent a promising approach to tumor though it

is still under investigation. For example, the incorporation of doxorubicin (DNR) in cancer cells

through the use of titanium dioxide whiskers can obviously increase the intracellular

concentration of DNR and enhance its potential anti-tumor efficiency. In view of these

researches, it is prudent to explore the possibility of ZnO nanoparticles loading chemotherapeutic

agent as a strategy in cancer therapy [12, 13]. To demonstrate this concept, 5-Fluorouracil (5-

FU), a frequently chemotherapeutic agent known to cause DNA damage and induce apoptosis

and cell death [14] was loaded on the ZnO NPs as drug delivery system. 5-FU, a pyrimidine

analog that interferes with thymidylate synthesis, has a broad spectrum of activity against solid

tumors. However, 5-FU has limitations that include a short biological half-life due to rapid

metabolism, incomplete and non-uniform oral absorption due to metabolism by di-hydro

perimidine dehydrogenase [15-18], toxic side effects on bone marrow and the gastrointestinal

tract, and non-selective action against healthy cells [19]. To prolong the circulation time of 5-FU

and increase its efficacy, numerous researchers have attempted to modify its delivery by use of

polymer conjugates or by incorporation of 5-FU into particulate carriers. The ultimate aim of

these strategies is to reduce 5-FU associated side effects and thereby improve its therapeutic

index [20-24].

In this study, we synthesized ZnO NPs using organic precursor method followed by the

encapsulation of 5-Fu to form ZnO NPs as a drug carrier. For its application analysis, their

physical characteristics, in vitro release behavior, and anti-tumor activity against MCF-7 was

investigated using various techniques (Viz: XRD, TEM, FESEM, FTIR, UV, HPLC, and flow

cytometry).

2. Experimental details

All the reagents grade chemicals and solvents were used without further purification and high

purity of 99.99%. 5, 6 dimethyl benzimidazole was purchased from Alfa Aesar England.

Zn(CH3COO)2.2H2O and 5-Fu were obtained from Sigma-Aldrich USA. Analytical grade

ethanol was obtained from Merck. Phosphate buffer solution (PBS)(0.1 M, pH 7.4) was prepared

by using double distilled water. To an ethanolic solution of 5, 6 dimethyl benzimidazole

(2.27mM, 500mg), 10 mL solution of zinc acetate dihydrate (Zn (Ac)2·2H2O) (4.54mM, 666 mg)
was added dropwise. The white color solution obtained was left for refluxing at 65°C for 4 hr

and cooled to room temperature. After removal of the solution, the requisite white precipitate

was obtained (81% yield). The organic impurities were removed via centrifugation for 10 min at

4000 rpm/min. The obtained white precipitate (Zinc compound) was washed with methanol and

dried in a Petri dish at room temperature. The zinc compound was subjected to calcination at 400

°C in a muffle furnace for 4hr, which resulted to ZnO NPs.

1.30 mg of drug5-Fu was dissolved in 10 µL of acetone and mixed with various molar ratios of

ZnO NPs (ZnONPs: 5-Fu = 100:1, 200:1 and 300:1). 1 mL of deionized water was added under

ultrasonic treatment for 30 min during which the acetone was evaporated. Centrifugation was

used to remove deionized water at 10000 rpm for 10 min to obtain drug loaded ZnO NPs, which

were dried with N2. The concentration of untrapped drug in water was measured by UV-vis

spectrophotometer at 204 and 266 nm which was used to calculate the encapsulation efficiency

(EE) and loading capacity (LC) of drug in ZnO NPs [25]. After encapsulation, 5-Fu encapsulated

ZnO NPs obtained in three forms D1, D2 and D3 with three different molar ratios 100:1, 200:1

and 300:1 (ZnO NPs: 5-FU) respectively.

Where: Q raw was the raw weight of drug used for encapsulation, Q unloaded was the weight of

the drug in ethanol, Q ZnO was the raw weight of ZnO used for drug entrapment. All the tests

were repeated thrice.

The release studies were performed using dialysis bag at pH 6.0 (tumor cell pH)and pH 7.4

(physiological pH). The 5-Fu loaded ZnO NPs (20 mg) was dispersed in PBS (pH 7.4, 5 mL) and
transferred to the dialysis bag. The dialysis bag was then immersed in 95 mL PBS of 6.0 and 7.4.

The release medium was continuously agitated with a stirrer at 100 rpm and 37 °C. At different

time intervals, 2 mL of the external medium was collected and replace with fresh PBS. HPLC

then determined the amount of released 5-Fu in the medium.

X-ray Diffraction analysis (XRD) was carried out using Rigaku Miniflex X-ray diffractometer

with Cu-Ka radiations (l =0.15406 nm) in the scan ranging from 20o to 80o of 2θ, with a step size

of 0.02o and scan speed of 2o/min. The morphology of the ZnO NPs was examined by field

emission scanning electron microscopy (FESEM) on Hitachi SU6600.The transmission electron

microscope, (TEM) images were using a JEOL JSM-2100F electron microscope (Tokyo, Japan)

operated at 200 kV. For the TEM analysis, specimen was prepared by dispersed it in ethanol and

a drop of the product was placed on carbon coated copper grids and dried before transferring it to

the TEM. FTIR was recorded as KBr pellets, using a Shimadzu IR Affinity-1 spectrometer with

a resolution of 400-4000 cm-1. UV–vis spectrophotometer (Thermo Spectronic 20 D+) between

190 and 600 nm wavelength, using quartz cuvettes examined the drug loading efficiency and

loading capacity.

Cell death was determined by staining with annexin V-FITC Apoptosis Detection Kit I (Jimmy

Biotech) and propidium iodide according to manufacturer's instructions [26]. The Cells

(MCF-7) were treated with D1, D2, and D3 at 10ppm concentration for 24h and washed with

PBS. Afterward, cells treated with (D1/D2/D3) were resuspended in binding buffer (300 μL).

Annexin V-FITC (5 μL) was added to the cells and incubated in the dark at room temperature for

15 min. They were further incubated for 5 min after adding propidium iodide (5 μL) and then

200 μL of cells treated with (D1/D2/D3) was added. Finally, the samples were examined by
FACS Calibur flow cytometry and data analysis was carried out with BD Cell Quest TM Pro

(version 5.2) software.

3. Results & discussion

Figure1 shows the typical XRD patterns of ZnO NPs and 5-Fu encapsulated NPs. It can be

clearly seen from both the XRD patterns that the ZnO NPs showed a single-phase nature with a

hexagonal wurtzite structure. There was no secondary phase detected, and the high intensity of

the peaks revealed the crystalline nature of the synthesized ZnO NPs. The XRD pattern of 5-Fu

encapsulated NPs is almost similar to ZnO NPs, indicating that the presence of 5-Fu does not

customize the crystal structure of the ZnO NPs, and successfully adsorbed on the surface of ZnO

NPs. The diffraction peaks of ZnO NPs and 5-Fu encapsulated NPs have been indexed to the

hexagonal wurtzite structured ZnO, which were well matched with that in JCPDS, 36-1451. The

crystallite sizes of the ZnO NPs and 5-Fu encapsulated NPs were estimated from X-ray line

broadening using Scherer's equation [27].

Where λ is the wavelength of X-ray radiation, and β is the full width at half maximum of the

peaks at the diffracting angle θ. Crystallite sizes were calculated, and found ~17 nm and~19nm

for ZnO NPs and 5-Fu encapsulated NPs, respectively. Refined values of the wurtzite lattice

parameters were found to be a=b=3.232Å, c=5.205Å for the ZnO NPs and a=b=3.241 Å,

c=5.202 Å for, and 5-Fu encapsulated NPs, respectively. These values are close to those of

lattice in the standard data (JCPDS, 36-1451). The increasing trend of lattice parameters

indicating that the volume of the unit cell for ZnO is significantly increased due to the adsorption

of 5-Fu on the surface of the ZnO NPs. In order to study the effect of addition of 5-Fu drug in

ZnO Matrix, analysis of the position of the XRD peak indicates that there is a shifting in peak's
position towards lower 2θ value, which resulted in the increase in interplanar spacing (d

value).The shifting of the peak's position clearly indicated that 5-Fu is incorporating into the

ZnO matrix. Additionally, the calculated lattice volume of 5-Fu incorporated ZnO NPs was

found to be higher (47.349 Å3) than that of pristine ZnO NPs (47.084 Å3). This increase in lattice

volume is due to the incorporation of 5-Fu, which lead to an expansion of the ZnO lattice. The

nearest-neighbor Zn-O bond length along the c-direction is given by [28].

√ ( )

where the internal parameter u in wurtzite structure is defined as.

( )

It should be noted that there is a strong correlation between the c/a ratio and u parameter. When

the c/a ratio decreases, u parameter increases in such a way that four tetrahedral distances remain

nearly constant through a distortion of tetrahedral angles due to long-range polar interactions

[29]. The observed values of c/a ratio and u parameters for pure ZnO NPs were 1.610 and 0.378

while for 5-Fu encapsulated ZnO NPs were 1.60 and 0.379, respectively. These results are in

close agreement with the standard relationship between c/a ratio and u parameters [30]. The

calculated Zn–O bond lengths for pure ZnO NPs was found to be 1.970 A, which is smaller than

that of the 5-Fu encapsulated ZnO NPs (1.973 A). It is clear that Zn–O bond lengths follow the

same increasing trend as lattice volume for 5-Fu encapsulated ZnO NPs. The changes in the

lattice volume, interplanar spacing, and bond length clearly indicated that the 5-Fu drug has been

successfully encapsulated to the ZnO crystal lattice.

Figure 2 shows the FESEM image of ZnO NPs and 5 Fu encapsulated ZnO NPs and their

corresponding EDX spectrum. Figure2 (a) depicts the FESEM images of ZnO NPs. It is evident

from the Figure (2 (a)) that the NPs have a spherical shape with an average size of ~ 19 nm.

Interestingly, some of the particles persist clear hexagon phase that can be easily seen from the

marked region in Figure 2 (a). The 5-Fu encapsulated ZnO sample also illustrated spherical

shape NPs with an increment in the size of ~ 22 nm. It is evident from the Figure (2(c)) that the

size of the ZnO NPs increased due to the encapsulation of 5-Fu. It might be because 5-Fu could

disturb the ZnO crystal lattice and resulted in the increased particle size. These observations are

in good agreement with the results obtained from XRD analysis. Additionally, the 5-

Fuparticlescan be clearly seen on the surface of ZnO NPs, which is also a good indicator of

successful encapsulation of 5-Fu on the surface of ZnO. The elemental composition of the ZnO

and 5-Fu encapsulated ZnO was determined by EDX analysis as shown in Figure. 2 (b) and 2(d),

respectively. The EDX measurements showed the presence of Zn and O peaks for ZnO (Figure2

(b), while the change in intensity of O and Zn peaks Figure 2(d) was observed which revealed

the encapsulation of 5-Fu on ZnO NPs.

The complementary morphological description is achieved through the TEM analysis. Figure 3

(a) shows the TEM image of ZnO NPs with an average size of ~18nm. It is evident from the

figure that the particles are spherical in shape and uniformly distributed. Figure 3(c) depicts the

TEM image of 5-Fu encapsulated ZnO NPs having the size of ~20 nm. The HRTEM image of

ZnO nanoparticles (Figure 3 (b)) shows well-resolved lattice spacing of 0.245 nm corresponding

to the d-spacing of the wurtzite ZnO (101) plane. The HRTEM image of 5-Fu encapsulated ZnO

NPs is shown in Figure 3 (d). The lattice spacing of the 5-Fu encapsulated ZnO NPs calculated

from the HRTEM image was found to increase to 0.247nm, and matches well with the lattice
spacing of (101) plane of ZnO. This increase in d-spacing of 5-Fu encapsulated ZnO NPs might

be due to the expansion of ZnO lattice during the encapsulation of 5-Fu in ZnO, which was also

revealed by XRD analysis. Therefore, TEM analysis in support of XRD and FESEM results

confirmed the successful encapsulation of 5-Fu on ZnO surface.

To study the change in Zn–O bonding due to the 5-Fu encapsulation, FTIR measurements of 5-

Fu encapsulated ZnO has been carried out. FTIR measurements were performed in the

wavenumber range 4000 to 400 cm-1 using KBr method at Room temperature as shown

inFigure4 (a). The FTIR spectra depicted main absorption bands near 3429 cm-1 represent O–H

mode, while at 2925 cm-1 indicating C–H mode, band arising from the absorption of atmospheric

CO2 on the metallic cations at 2216 cm-1and 1400–1600 cm-1are due to C=O stretching mode.

The absorption band at 437 cm-1 is the stretching mode of ZnO 30. However, in case of 5-Fu

encapsulated NPs Fig. 4(b) the bands at 1471 and 1605 cm-1 correspond to C=N, and C=C of 5-

Fu, respectively and the value of absorption bands were found to be an increase of 438 cm-1. The

change in the peak position of ZnO absorption bands reflects that Zn–O–Zn network is perturbed

by the presence of 5-Fu in its vicinity. Therefore, the FTIR results also suggested that

encapsulation of 5-Fu on ZnO matrix as observed in XRD measurements.

A brief mechanism for the formation of ZnO-NPs is as follows: Under the above experimental

conditions, we may hypothesized that the hydrogen bond between CH3 (alkyl group)of 5, 6

dimethyl benzimidazole reacts with the acetate group of zinc acetate to form the zinc compound

(C9H10N2Zn)and ethyl-ethanoate (C2H5COOCH3) (Eq. (1)). An increase in the reflux time of this

solution increases the amount of solvent (EtOH) that directly reacts with the formed compound

(C9H10N2Zn) to form Zn(OH)2and regenerate 5, 6 dimethyl benzimidazole(Eq.(2)). Zinc

hydroxide ions exist in the solution as the ionic forms Zn2+and 2OH- (Eq. (3)). At a higher
temperature, the zinc and hydroxyl (OH−) ions changed into pure zinc oxide (ZnO) and water

(Eq. (4)). The by-product water molecule from the reaction is leached out during calcination

process [31]. It is assumed that the initially formed Zn(OH)2 in the solution, resulted in the

aggregation of nuclei and that the acquisition of sufficient thermal energy from the reflux pot

leads to the formation of small active molecules of ZnO. These initial or active ZnO

nuclei/particles are expected to be the building blocks for the formation of the ZnO NPs [32].

C2H5OH
Zn(CH3COO)2.2H2O + C9H10N2 C9H10N2Zn + 2C2H5COOCH3 ---------(1)

C9H10N2Zn + 2C2H5COOCH3 C9H10N2 + Zn(OH)2 ---------(2)

---------(3)
Zn(OH)2 Zn2+ + 2OH
---------(4)
Zn2+ + 2OH ZnO + H2O

The encapsulation efficiency and loading efficiency of 5-Fu loaded ZnO NPs were assessed and

calculated as 85.7% and 4.5%, respectively. The results indicated that ZnO NPs encapsulated

with 5-Fu may efficiently act as the anticancer drug delivery carrier. The release of drug

molecules depends upon the pH of the medium and releasing time Figure 5.The drug release at

pH 7.4 was slow and sustained, with a releasing ratio of about 17% within 36 h. However, at

lower pH 6.0, the 5-Fu release rate was much faster, with approximately 69% within 36 h as

shown in Figure 5. These results clearly suggested that the drug release was slow at

physiological pH and resulted in the lower toxicity to the normal cells. Moreover, at pH 6.0

(where tumor cell is metabolically active), the release of 5-Fu from encapsulated ZnO-NPs is

more significant and thus increases bioavailability of 5-Fu towards cancer cell, therefore future

research warranted target killing of tumor cell.

With the purpose of detecting mechanism of 5-Fu encapsulated ZnO NPs induced cellular death

(necrosis or apoptosis), the flow cytometry experiment were performed on encapsulated 5-Fu
ZnO-NPs (D1, D2 and D3) with three different molar ratios 100:1, 200:1 and 300:1 (ZnO NPs:

5-FU) respectively. These three forms were treated to MCF-7 cell line with 10 ppm

concentration for 24 hr, and the results were shown in Figure 6. Four areas in the diagrams stand

for necrotic cells (positive for PI and negative for annexin/FITC, left square on the top), live cells

(negative for annexin and PI, left square at the bottom), late apoptosis or necrosis cells (positive

for annexin and PI, right square on the top) and apoptosis cells (negative for PI and positive for

annexin, right square at the bottom), respectively. Figure 6, illustrated the cytotoxicity details of

MCF-7 with different molar ratio of 5-Fu encapsulated ZnO-NPs, D3 at 10 ppm showed the

highest population of apoptotic cells (56.06 %),Whereas D2 and D1 at the same concentration

resulted in the decrease of cell death to 37.1% and 30.55% respectively. Moreover, flow

cytometry analysis of the individual component of D3 resulted in 11.81% cell death induced by

5-Fu while the ZnO fraction caused 31.51% cell death. The increase in the cell death seems to be

dependent on the ZnO ratio. The rise in the ZnO ratio (from 100 to 200 to 300mM) produced a

corresponding increase in the 5-Fu-induced cell death that is approximately five fold higher than

5-Fu and 1.7fold higher than ZnO NPs. Thus, illustrating encapsulation of 5-Fu on ZnO-NPs

enhances the cytotoxicity (56.06% cell death) of cancer cell in comparison to 5-Fluorouracil.

4. Conclusion

To summarize, in this study we have successfully synthesized and characterized ZnO NPs by

a simple, cost effective and biocompatible organic precursor by using various techniques

such as XRD, FESEM, TEM, and FTIR spectroscopy. Notably, encapsulation of 5-

Fluorouracil were prepared and characterized using above-mentioned standard techniques.

Moreover, we had tried to explore the potential application of encapsulated ZnO NPs for the

delivery of anticancer drug 5-Fu over tumor cells. The results demonstrated that the
 encapsulated 5-Fu in the matrix of ZnO NPs can transport more anti-cancer drug (5-Fu) for

internalization and increase the bioavailability of 5-Fu in MCF-7 cells and thus more toxicity

to the cancer cell. Thus, encapsulation could induce significant enhancement in anti-tumor

activity. Therefore, ZnO NPs present a new drug delivery property and a promising strategy

of encapsulation for comprehensive cancer treatment.

Acknowledgement

The author would like to extend their sincere appreciation to the Deanship of Scientific

Research at King Saud University, Riyadh, Saudi Arabia for its funding this research group

No. RGP-150.

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